Nucleotide sequences and corresponding polypeptides conferring modulated growth rate and biomass in plants grown in saline conditions

ABSTRACT

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able confer the trait of improved plant size, vegetative growth, growth rate, seedling vigor and/or biomass in plants challenged with saline conditions. The present invention further relates to the use of these nucleic acid molecules and polypeptides in making transgenic plants, plant cells, plant materials or seeds of a plant having plant size, vegetative growth, growth rate, seedling vigor and/or biomass that are improved in saline conditions with respect to wild-type plants grown under similar conditions.

This application is a Division of co-pending application Ser. No.12/282,342 filed on Nov. 17, 2008 and for which priority is claimedunder 35 U.S.C. § 120. Application Ser. No. 12/282,342 is a NationalPhase under 35 U.S.C. § 371 of PCT International Application No.PCT/US2007/06544 which has the International filing date of Mar. 14,2007, which designated the United States of America, and which claimspriority under 35 U.S.C. § 119(e) of U.S. Provisional Application No.60/782,735, filed Mar. 14, 2006. The entire contents of each of whichare hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acid molecules andtheir corresponding encoded polypeptides able to enhance plant growthunder saline conditions. The present invention further relates to usingthe nucleic acid molecules and polypeptides to make transgenic plants,plant cells, plant materials or seeds of a plant having improved growthrate, vegetative growth, seedling vigor and/or biomass under salineconditions as compared to wild-type plants grown under similarconditions.

BACKGROUND OF THE INVENTION

Plants specifically improved for agriculture, horticulture, biomassconversion, and other industries (e.g. paper industry, plants asproduction factories for proteins or other compounds) can be obtainedusing molecular technologies. As an example, great agronomic value canresult from enhancing plant growth in saline conditions.

A wide variety agriculturally important plant species demonstratesignificant sensitivity to saline water and/or soil. Upon saltconcentration exceeding a relatively low threshold, many plants sufferfrom stunted growth, necrosis, and death that results in an overallstunted appearance and reduced yields of plant material, seeds, fruitand other valuable products. Physiologically, plants challenged withsalinity experience disruption in ion and water homeostasis, inhibitionof metabolism, and damage to cellular membranes that result indevelopmental arrest and cell death (Huh et al. (2002) Plant J,29(5):649-59).

In many of the world's most productive agricultural regions,agricultural activities themselves lead to increased water and soilsalinity, which threatens their sustained productivity. One example iscrop irrigation in arid regions that have abundant sunlight. Afterirrigation water is applied to cropland, it is removed by the processesof evaporation and evapotranspiration. While these processes removewater from the soil, they leave behind dissolved salts carried inirrigation water. Consequently, soil and groundwater salt concentrationsbuild over time, rendering the land and shallow groundwater saline andthus damaging to crops.

In addition to human activities, natural geological processes havecreated vast tracts of saline land that would be highly productive ifnot saline. In total, approximately 20% of the irrigated lands in arenegatively affected by salinity. (Yamaguchi and Blumwald, 2005, Trendsin Plant Science, 10: 615-620). For these and other reasons, it is ofgreat interest and importance to identify genes that confer improvedsalt tolerance characteristics to thereby enable one to createtransgenic plants (such as crop plants) with enhanced growth and/orproductivity characteristics in saline conditions.

The availability and sustainability of a stream of food and feed forpeople and domesticated animals has been a high priority throughout thehistory of human civilization and lies at the origin of agriculture.Specialists and researchers in the fields of agronomy science,agriculture, crop science, horticulture, and forest science are eventoday constantly striving to find and produce plants with an increasedgrowth potential to feed an increasing world population and to guaranteea supply of reproducible raw materials. The robust level of research inthese fields of science indicates the level of importance leaders inevery geographic environment and climate around the world place onproviding sustainable sources of food, feed and energy.

Manipulation of crop performance has been accomplished conventionallyfor centuries through selection and plant breeding. The breeding processis, however, both time-consuming and labor-intensive. Furthermore,appropriate breeding programs must be specially designed for eachrelevant plant species.

On the other hand, great progress has been made in using moleculargenetic approaches to manipulate plants to provide better crops. Throughthe introduction and expression of recombinant nucleic acid molecules inplants, researchers are now poised to provide the community with plantspecies tailored to grow more efficiently and yield more product despitesuboptimal geographic and/or climatic environments. These new approacheshave the additional advantage of not being limited to one plant species,but instead being applicable to multiple different plant species (Zhanget al. (2004) Plant Physiol. 135:615; Zhang et al. (2001) Proc. Natl.Acad. Sci. USA 98:12832).

Despite this progress, today there continues to be a great need forgenerally applicable processes that improve forest or agricultural plantgrowth to suit particular needs depending on specific environmentalconditions. To this end, the present invention is directed toadvantageously manipulating plant tolerance to salinity in order tomaximize the benefits of various crops depending on the benefit sought,and is characterized by expression of recombinant DNA molecules inplants. These molecules may be from the plant itself, and simplyexpressed at a higher or lower level, or the molecules may be fromdifferent plant species.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated nucleic acidmolecules and polypeptides and their use in making transgenic plants,plant cells, plant materials or seeds of plants having improved growthcharacteristics in saline conditions compared to wild-type plants undersimilar or identical conditions.

The present invention also relates to processes for increasing thegrowth potential of plants challenged with saline conditions due to salttolerance derived from recombinant nucleic acid molecules andpolypeptides. The phrase “increasing growth potential” refers tocontinued growth in saline conditions, better yield after exposure tosaline conditions and/or increased vigor in saline conditions.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Amino acid sequence alignment of homologues of Ceres clone 8686(SEQ ID NO: 80). Conserved regions are enclosed in a box.

FIG. 2. Amino acid sequence alignment of homologues of Ceres clone375578 (SEQ ID NO: 252). Conserved regions are enclosed in a box.

FIG. 3 Amino acid sequence alignment of homologues of Ceres clone 105319(SEQ ID NO: 106). Conserved regions are enclosed in a box.

FIG. 4 Amino acid sequence alignment of homologues of Ceres clone 29658(SEQ ID NO: 123). Conserved regions are enclosed in a box.

FIG. 5 Amino acid sequence alignment of homologues of Ceres clone 2767(SEQ ID NO: 132). Conserved regions are enclosed in a box.

FIG. 6 Amino acid sequence alignment of homologues of Ceres clone 16403(SEQ ID NO: 146). Conserved regions are enclosed in a box.

FIG. 7 Amino acid sequence alignment of homologues of Ceres clone 3964(SEQ ID NO: 154). Conserved regions are enclosed in a box.

FIG. 8 Amino acid sequence alignment of homologues of Ceres clone 965405(SEQ ID NO: 172). Conserved regions are enclosed in a box.

FIG. 9 Amino acid sequence alignment of a conserved region of Ceresclones 375578 (SEQ ID NO: 306) and 335348 (SEQ ID NO: 304) andhomologues. Conserved regions are enclosed in a box.

DETAILED DESCRIPTION OF THE INVENTION 1. The Invention

The present invention discloses novel isolated nucleic acid molecules,nucleic acid molecules that interfere with these nucleic acid molecules,nucleic acid molecules that hybridize to these nucleic acid molecules,and isolated nucleic acid molecules that encode the same protein due tothe degeneracy of the DNA code. Additional embodiments of the presentapplication further include the polypeptides encoded by the isolatednucleic acid molecules of the present invention.

More particularly, the nucleic acid molecules of the present inventioncomprise: (a) a nucleotide sequence that encodes an amino acid sequenceand that is at least 85% identical to any one of SEQ ID Nos. 80, 99,106, 123, 132, 146, 154 and 172 respectively, (b) a nucleotide sequencethat is complementary to any one of the nucleotide sequences accordingto (a), (c) a nucleotide sequence according to any one of SEQ ID NOs.79, 98, 105, 122, 131, 145, 153 and 171, (d) a nucleotide sequence ableto interfere with any one of the nucleotide sequences according to (a),(e) a nucleotide sequence able to form a hybridized nucleic acid duplexwith the nucleic acid according to any one of paragraphs (a)-(d) at atemperature from about 5° C. to about 10° C. below a melting temperatureof the hybridized nucleic acid duplex, (f) a nucleotide sequenceencoding any one of amino acid sequences of SEQ ID NOS. 80, 99, 106,123, 132, 146, 154 and 172, (g) a nucleotide sequence encoding any oneof the amino acid sequences with an HMM bit score greater than 20 thatfits an HMM based on the sequences aligned in any one of FIGS. 1-8, and(h) a nucleotide sequence encoding an amino acid sequence having afragment that fits an HMM based on the sequences aligned in FIG. 9 andwhich has an HMM bit score greater than 400.

Additional embodiments of the present invention include thosepolypeptide and nucleic acid molecule sequences disclosed in SEQ ID NOs:81-97, 100-104, 107-121, 124-130, 133-144, 147-152, 155-170, 173-252 and269-315.

The present invention further embodies a vector comprising a firstnucleic acid having a nucleotide sequence encoding a plant transcriptionand/or translation signal, and a second nucleic acid having a nucleotidesequence according to the isolated nucleic acid molecules of the presentinvention. More particularly, the first and second nucleic acids may beoperably linked. Even more particularly, the second nucleic acid may beendogenous to a first organism, and any other nucleic acid in the vectormay be endogenous to a second organism. Most particularly, the first andsecond organisms may be different species.

In a further embodiment of the present invention, a host cell maycomprise an isolated nucleic acid molecule according to the presentinvention. More particularly, the isolated nucleic acid molecule of thepresent invention found in the host cell of the present invention may beendogenous to a first organism and may be flanked by nucleotidesequences endogenous to a second organism. Further, the first and secondorganisms may be different species. Even more particularly, the hostcell of the present invention may comprise a vector according to thepresent invention, which itself comprises nucleic acid moleculesaccording to those of the present invention.

In another embodiment of the present invention, the isolatedpolypeptides of the present invention may additionally comprise aminoacid sequences that are at least 85% identical to any one of SEQ ID Nos.80, 99, 106, 123, 132, 146, 154 and 172.

Other embodiments of the present invention include methods ofintroducing an isolated nucleic acid of the present invention into ahost cell. More particularly, an isolated nucleic acid molecule of thepresent invention may be contacted to a host cell under conditionsallowing transport of the isolated nucleic acid into the host cell. Evenmore particularly, a vector as described in a previous embodiment of thepresent invention may be introduced into a host cell by the same method.

Methods of detection are also available as embodiments of the presentinvention. Particularly, methods for detecting a nucleic acid moleculeaccording to the present invention in a sample. More particularly, theisolated nucleic acid molecule according to the present invention may becontacted with a sample under conditions that permit a comparison of thenucleotide sequence of the isolated nucleic acid molecule with anucleotide sequence of nucleic acid in the sample. The results of suchan analysis may then be considered to determine whether the isolatednucleic acid molecule of the present invention is detectable andtherefore present within the sample.

A further embodiment of the present invention comprises a plant, plantcell, plant material or seeds of plants comprising an isolated nucleicacid molecule and/or vector of the present invention. More particularly,the isolated nucleic acid molecule of the present invention may beexogenous to the plant, plant cell, plant material or seed of a plant.

A further embodiment of the present invention includes a plantregenerated from a plant cell or seed according to the presentinvention. More particularly, the plant, or plants derived from theplant, plant cell, plant material or seeds of a plant of the presentinvention preferably has increased size (in whole or in part), increasedvegetative growth and/or increased biomass (sometimes hereinaftercollectively referred to as increased biomass) in saline conditions, ascompared to a wild-type plant cultivated under identical conditions.Furthermore, the transgenic plant may comprise a first isolated nucleicacid molecule of the present invention, which encodes a protein involvedin improving growth and phenotype characteristics in saline conditions,and a second isolated nucleic acid molecule which encodes a promotercapable of driving expression in plants, wherein the growth andphenotype improving component and the promoter are operably linked. Morepreferably, the first isolated nucleic acid may be mis-expressed in thetransgenic plant of the present invention, and the transgenic plantexhibits improved characteristics as compared to a progenitor plantdevoid of the polynucleotide, when the transgenic plant and theprogenitor plant are cultivated under identical, saline conditions. Inanother embodiment of the present invention the improved growth andphenotype characteristics may be due to the inactivation of a particularsequence, using for example an interfering RNA.

A further embodiment consists of a plant, plant cell, plant material orseed of a plant according to the present invention which comprises anisolated nucleic acid molecule of the present invention, wherein theplant, or plants derived from the plant, plant cell, plant material orseed of a plant, has the improved growth and phenotype characteristicsin saline conditions as compared to a wild-type plant cultivated underidentical conditions.

The polynucleotide conferring increased biomass or vigor in salineconditions may be mis-expressed in the transgenic plant of the presentinvention, and the transgenic plant exhibits an increased biomass orvigor as compared to a progenitor plant devoid of the polynucleotide,when the transgenic plant and the progenitor plant are cultivated underidentical saline conditions. In another embodiment of the presentinvention increased biomass or vigor phenotype may be due to theinactivation of a particular sequence, using for example an interferingRNA.

Another embodiment consists of a plant, plant cell, plant material orseed of a plant according to the present invention which comprises anisolated nucleic acid molecule of the present invention, wherein theplant, or plants derived from the plant, plant cell, plant material orseed of a plant, has increased biomass or vigor in saline conditions ascompared to a wild-type plant cultivated under identical conditions.

Another embodiment of the present invention includes methods ofenhancing biomass or vigor in plants challenged with saline conditions.More particularly, these methods comprise transforming a plant with anisolated nucleic acid molecule according to the present invention.Preferably, the method is a method of enhancing biomass or vigor in thetransgenic plant, whereby the plant is transformed with a nucleic acidmolecule encoding the polypeptide of the present invention.

Polypeptides of the present invention include sequences belonging to theconsensus sequence families shown in FIGS. 1-9 as delineated by HiddenMarkov Models (HMMs).

2. Definitions

The following terms are utilized throughout this application:

Functionally Comparable Proteins or Functional Homologs: This phrasedescribes a set of proteins that perform similar functions within anorganism. By definition, perturbation of an individual protein withinthat set (through misexpression or mutation, for example) is expected toconfer a similar phenotype as compared to perturbation of any otherindividual protein. Such proteins typically share sequence similarityresulting in similar biochemical activity. Within this definition,homologs, orthologs and paralogs are considered to be functionallycomparable.

Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily the same, degree.Typically, comparable proteins give the same characteristics where thequantitative measurement due to one of the comparables is at least 20%of the other; more typically, between 30 to 40%; even more typically,between 50-60%; even more typically between 70 to 80%; even moretypically between 90 to 100% of the other.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if other filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Hidden Markov Model (HMM): HMM is a statistical description of asequence family's consensus. The model is indicative of similarity of apolypeptide sequence to a group of structurally and functionally relatedpolypeptides (Durbin, R., Eddy, S. R., Krogh, A. & Mitchison, G. J.Biological Sequence Analysis: Probabilistic Models of Proteins andNucleic Acids Cambridge University Press, Cambridge UK, 1998).

HMM based on specified sequences: An HMM profile based on specifiedsequences is the output model generated by the program HMMER 2.3.2(released Oct. 3, 2003 under a GNU general public license, and availablefrom various sources, such as the HMMER website on the internet)configured with default parameters, the model being built by the programusing as input the specified sequences. The program outputs the model asa text file.

HMM bit score: An HMM bit score is a probabilistic indication ofconfidence that a sequence fits the model. The bit score reflectswhether the sequence is a better fit to an HMM of interest than to anull model of nonhomologous sequences. A significant HMM bit score isgreater than zero, but is typically greater than 20. The HMM bit scoreof a polypeptide sequence fitted to an HMM profile can be determined byfitting the polypeptide to the HMM with program HMMER 2.3.2 configuredfor glocal alignments.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome, including a gene or coding region from a different plantspecies or from a non-plant organism.

Percentage of sequence identity: As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence and a subject sequence. A subject sequence typically hasa length that is from about 80 percent to 250 percent of the length ofthe query sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105,110, 115, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, or 250 percent of the length of the query sequence. A query nucleicacid or amino acid sequence is aligned to one or more subject nucleicacid or amino acid sequences using the computer program ClustalW(version 1.83, default parameters), which allows alignments of nucleicacid or protein sequences to be carried out across their entire length(global alignment). Chenna et al. (2003) Nucleic Acids Res.31(13):3497-500.

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For an alignment of multiple nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launcherwebsite and at the European Bioinformatics Institute website on theWorld Wide Web.

To determine a percent identity for polypeptide or nucleic acidsequences between a query and a subject sequence, the sequences arealigned using Clustal W and the number of identical matches in thealignment is divided by the query length, and the result is multipliedby 100. The output is the percent identity of the subject sequence withrespect to the query sequence. It is noted that the percent identityvalue can be rounded to the nearest tenth. For example, 78.11, 78.12,78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17,78.18, and 78.19 are rounded up to 78.2.

Photosynthetic efficiency: photosynthetic efficiency, or electrontransport via photosystem II, is estimated by the relationship betweenFm, the maximum fluorescence signal and the variable fluorescence, Fv.Here, a reduction in the optimum quantum yield (Fv/Fm) indicates stressand can be used to monitor the performance of transgenic plants comparedto non-transgenic plants under salt stress conditions.

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns. Regulatory regions can beclassified in two categories, promoters and other regulatory regions.

Salt tolerance: Plant species vary in their capacity to toleratesalinity. “Salinity” can be defined as the set of environmentalconditions under which a plant will begin to suffer the effects ofelevated salt concentration, such as ion imbalance, decreased stomatalconductance, decreased photosynthesis, decreased growth rate, increasedcell death, loss of turgor (wilting), or ovule abortion. For thesereasons, plants experiencing salinity stress typically exhibit asignificant reduction in biomass and/or yield.

Elevated salinity may be caused by natural, geological processes and byhuman activities, such as irrigation. Since plant species vary in theircapacity to tolerate water deficit, the precise environmental saltconditions that cause stress cannot be generalized. However, undersaline conditions, salt tolerant plants produce higher biomass, yieldand survivorship than plants that are not salt tolerant. Differences inphysical appearance, recovery and yield can be quantified andstatistically analyzed using well known measurement and analysismethods.

Seedling area: The total leaf area of a young plant about 2 weeks old.

Seedling vigor: As used herein, “seedling vigor” refers to the plantcharacteristic whereby the plant emerges from soil faster, has anincreased germination rate (i.e., germinates faster), has faster andlarger seedling growth and/or germinates faster under salt conditions ascompared to the wild-type or control under similar conditions. Seedlingvigor has often been defined to comprise the seed properties thatdetermine “the potential for rapid, uniform emergence and development ofnormal seedlings under a wide range of field conditions”.

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)−5° C. to T_(m)−10° C. Medium or moderate stringencyconditions are those providing T_(m)−20° C. to T_(m)−29° C. Lowstringency conditions are those providing a condition of T_(m)−40° C. toT_(m)−48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:

T _(m)=81.5-16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (I)

where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L 0.63(%formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid(Bonner et al. (1973) J. Mol. Biol. 81:123). The T_(m) of Equation II isaffected by the nature of the hybrid: for DNA-RNA hybrids, T_(m) is10-15° C. higher than calculated; for RNA-RNA hybrids, T_(m) is 20-25°C. higher. Because the T_(m) decreases about 1° C. for each 1% decreasein homology when a long probe is used (Frischauf et al. (1983) J. MolBiol, 170: 827-842), stringency conditions can be adjusted to favordetection of identical genes or related family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present invention are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

For example, the hybridization step may be performed in aqueoushybridization solution at a temperature between 63° C. and 70° C., morepreferably at a temperature between 65° C. and 68° C. and mostpreferably at a temperature of 65° C. Alternatively, the high stringencyhybridization step may be performed in formamide hybridization solutionat a temperature between 40° C. and 46° C., at a temperature between 41°C. and 44° C. and most preferably at a temperature of 42° C.

A wash step follows hybridization, and an initial wash is performed withwash solution 1 at 25° C. or 37° C. Following the initial wash,additional washes are performed with wash solution 1 at a temperaturebetween 63° C. and 70° C., more preferably at a temperature between 65°C. and 68° C. and most preferably at a temperature of 65° C. The numberof additional wash steps can be 1, 2, 3, 4, 5 or more. The time of boththe initial and additional wash steps may be 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 1.5hours, 2 hours or more.

Set forth below are the composition of the hybridization and washsolutions and their components. A person of ordinary skill in the artwill recognize that these solutions are typical and exemplary of highstringency hybridization solutions.

Aqueous Hybridization Solution: 6×SSC or 6×SSPE

-   -   0.05% Blotto or 5×Denhardt's Reagent    -   100 μg/ml denatured salmon sperm DNA    -   0.05% SDS

Formamide Hybridization Solution: 50% Formamide

-   -   6×SSC or 6×SSPE    -   0.05% Blotto or 5×Denhardt's Reagent    -   100 μg/ml denatured salmon sperm DNA    -   0.05% SDS

Wash Solution 1: 2×SSC or SSPE

-   -   0.1% SDS

Wash Solution 2: 0.1×SSC or SSPE

-   -   0.5% SDS

20×SSC: 175.3 g NaCl

-   -   88.2 g Sodium Citrate    -   Bring to 800 ml with H₂O    -   Adjust to pH 7 with 10 n NaOH    -   Bring to 1L with H₂O

20×SSPE: 175.3 g NaCl

-   -   27.6 g NaH₂PO₄    -   Bring to 800 ml with H₂O•H₂O    -   7.4 g EDTA    -   Adjust to pH 7.4 with 10 n NaOH    -   Bring to 1L with H₂O        1×BLOTTO: 5% Non-fat dry milk    -   0.02% Sodium azide

50×Denhardts's Reagent: 5 g Ficoll

-   -   5 g Polyvinylpyrrolidone    -   5 g BSA    -   Adjust to 500 ml with H₂O

Superpool: As used in the context of the current invention, a“superpool” contains an equal amount of seed from 500 different events,representing 100 distinct exogenous nucleotide sequences. An event is aplant carrying a unique insertion of a distinct exogenous sequence whichmisexpresses that sequence. Transformation of a single polynucleotidesequence can result in multiple events because the sequence can insertin a different part of the genome with each transformation.

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the T₀ plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

T₄: As used in the current application, the term T₄ refers to thirdgeneration progeny of the plant that is the direct result of atransformation experiment. T₄ progeny are the result ofself-fertilization or cross pollination of a T₃ plant.

Transformation: Examples of means by which this can be accomplished aredescribed below and include Agrobacterium-mediated transformation (ofdicots (Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson andLipman (1988) Proc. Natl. Acad. Sci. (USA) 85: 2444), of monocots(Yamauchi et al. (1996) Plant Mol Biol. 30:321-9; Xu et al. (1995) PlantMol. Biol. 27:237; Yamamoto et al. (1991) Plant Cell 3:371), andbiolistic methods (P. Tijessen, “Hybridization with Nucleic Acid Probes”In Laboratory Techniques in Biochemistry and Molecular Biology, P. C.vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam), electroporation,in planta techniques and the like. Such a plant containing the exogenousnucleic acid is referred to here as a T₀ for the primary transgenicplant and T₁ for the first generation.

3. Important Characteristics of the Polynuceotides and Polypeptides ofthe Invention

The nucleic acid molecules and polypeptides of the present invention areof interest because when the nucleic acid molecules are mis-expressed(i.e., when expressed at a non-natural location or in an increased ordecreased amount relative to wild-type) they produce plants that exhibitimproved salt tolerance as compared to wild-type plants, as evidenced bythe results of various experiments disclosed below. In particular,plants transformed with at least one of the nucleic acid molecules andpolypeptides of the present invention have increased salt growth indexvalues as compared to wild-type plants. For example, plants transformedwith the sequences of the present invention can exhibit increases in SGIvalues of at least 25%, at least 50%, at least 75%, at least 100%, atleast 200%, at least 300%, at least 400%, or even at least 500%. Thistrait can be used to exploit or maximize plant products. For example,the nucleic acid molecules and polypeptides of the present invention areused to increase the expression of genes that cause the plant to haveimproved biomass, growth rate and/or seedling vigor in salineconditions.

Because the disclosed sequences and methods increase vegetative growthand growth rate in saline conditions, the disclosed methods can be usedto enhance plant growth in plants irrigated with saline water and/orgrown in saline soil. For example, plants of the invention show, undersaline conditions, increased photosynthetic efficiency and increasedseedling area as compared to a plant of the same species that is notgenetically modified for substantial vegetative growth. Examples ofincreases in biomass production include increases of at least 5%, atleast 20%, or even at least 50%, when compared to an amount of biomassproduction by a wild-type plant of the same species under identicalconditions.

Seed or seedling vigor is an important characteristic that can greatlyinfluence successful growth of a plant, such as crop plants. Adverseenvironmental conditions, such as saline conditions, can affect a plantgrowth cycle, germination of seeds and seedling vigor (i.e. vitality andstrength under such conditions can differentiate between successful andfailed crop growth). Seedling vigor has often been defined to comprisethe seed properties that determine “the potential for rapid, uniformemergence and development of normal seedlings under a wide range offield conditions”. Hence, it would be advantageous to develop plantseeds with increased vigor, particularly in elevated salinity.

For example, increased seedling vigor would be advantageous for cerealplants such as rice, maize, wheat, etc. production. For these crops,germination and growth can often be slowed or stopped by salination.Genes associated with increased seed vigor and/or salination tolerancehave therefore been sought for producing improved crop varieties. (Waliaet al. (2005) Plant Physiology 139:822-835).

4. The Polypeptides/Polynucleotides of the Invention

The polynucleotides of the present invention and the proteins expressedvia translation of these polynucleotides are set forth in the SequenceListing, specifically SEQ ID NOs. 79-253 and 269-315. The SequenceListing also consists of functionally comparable proteins. Polypeptidescomprised of a sequence belonging to the consensus sequence familiesshown in FIGS. 1 to 9 as delineated by HMMs can be utilized for thepurposes of the invention, namely to make transgenic plants withimproved biomass, growth rate and/or seedling vigor in salineconditions.

5. Use of the Polypeptides to Make Transgenic Plants

To use the sequences of the present invention or a combination of themor parts and/or mutants and/or fusions and/or variants of them,recombinant DNA constructs are prepared that comprise the polynucleotidesequences of the invention inserted into a vector and that are suitablefor transformation of plant cells. The construct can be made usingstandard recombinant DNA techniques (see, Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, 1989, New York.) and can be introduced into the plantspecies of interest by, for example, Agrobacterium-mediatedtransformation, or by other means of transformation, for example, asdisclosed below.

The vector backbone may be any of those typically used in the field suchas plasmids, viruses, artificial chromosomes, BACs, YACs, PACs andvectors such as, for instance, bacteria-yeast shuttle vectors, lambdaphage vectors, T-DNA fusion vectors and plasmid vectors (see, Shizuya etal. (1992) Proc. Natl. Acad. Sci. USA 89: 8794-8797; Hamilton et al.(1996) Proc. Natl. Acad. Sci. USA 93: 9975-9979; Burke et al. (1987)Science 236:806-812 ; Sternberg N. et al. (1990) Proc Natl Acad Sci U SA. 87:103-7; Bradshaw et al. (1995) Nucl Acids Res 23: 4850-4856;Frischauf et al. (1983) J. Mol Biol 170: 827-842; Huynh et al., Glover NM (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press(1985); Walden et al. (1990) Mol Cell Biol 1: 175-194).

Typically, the construct comprises a vector containing a nucleic acidmolecule of the present invention with any desired transcriptionaland/or translational regulatory sequences such as, for example,promoters, UTRs, and 3′ end termination sequences. Vectors may alsoinclude, for example, origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, and introns. The vectormay also comprise a marker gene that confers a selectable phenotype onplant cells. The marker may preferably encode a biocide resistancetrait, particularly antibiotic resistance, such as resistance to, forexample, kanamycin, bleomycin, or hygromycin, or herbicide resistance,such as resistance to, for example, glyphosate, chlorosulfuron orphosphinotricin.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, more than one regulatory region can be operably linkedto said sequence.

To “operably link” a promoter sequence to a sequence, the translationinitiation site of the translational reading frame of said sequence istypically positioned between one and about fifty nucleotides downstreamof the promoter. A promoter can, however, be positioned as much as about5,000 nucleotides upstream of the translation initiation site, or about2,000 nucleotides upstream of the transcription start site. A promotertypically comprises at least a core (basal) promoter. A promoter alsomay include at least one control element, such as an enhancer sequence,an upstream element or an upstream activation region (UAR). For example,a suitable enhancer is a cis-regulatory element (−212 to −154) from theupstream region of the octopine synthase (ocs) gene. Fromm et al. (1989)Plant Cell 1:977-984.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. For example, a promoter that is activepredominantly in a reproductive tissue (e.g., fruit, ovule, pollen,pistils, female gametophyte, egg cell, central cell, nucellus,suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo,zygote, endosperm, integument, or seed coat) can be used. Thus, as usedherein a cell type- or tissue-preferential promoter is one that drivesexpression preferentially in the target tissue, but may also lead tosome expression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et al. (1989) Plant Cell 1:855-866; Bustos et al. (1989) PlantCell 1:839-854; Green et al. (1988) EMBO J. 7: 4035-4044; Meier et al.(1991) Plant Cell 3: 309-316; and Zhang et al. (1996) Plant Physiology110: 1069-1079.

Examples of various classes of promoters are described below. Some ofthe promoters indicated below are described in more detail in U.S.patent application Ser. Nos. 60/505,689; 60/518,075; 60/544,771;60/558,869; 60/583,691; 60/619,181; 60/637,140; 10/950,321; 10/957,569;11/058,689; 11/172,703; 11/208,308; and PCT/US05/23639. It will beappreciated that a promoter may meet criteria for one classificationbased on its activity in one plant species, and yet meet criteria for adifferent classification based on its activity in another plant species.

Other Regulatory Regions: A 5′ untranslated region (UTR) can be includedin nucleic acid constructs described herein. A 5′ UTR is transcribed,but is not translated, and lies between the start site of the transcriptand the translation initiation codon and may include the +1 nucleotide.A 3′ UTR can be positioned between the translation termination codon andthe end of the transcript. UTRs can have particular functions such asincreasing mRNA stability or attenuating translation. Examples of 3′UTRs include, but are not limited to, polyadenylation signals andtranscription termination sequences, e.g., a nopaline synthasetermination sequence.

Various promoters can be used to drive expression of the polynucleotidesof the present invention. Nucleotide sequences of such promoters are setforth in SEQ ID NOS: 1-78. Some of them can be broadly expressingpromoters, others may be more tissue preferential.

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues or plantcells. For example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of theshoot, shoot tip (apex), and leaves, but weakly or not at all in tissuessuch as roots or stems. As another example, a broadly expressingpromoter can promote transcription of an operably linked sequence in oneor more of the stem, shoot, shoot tip (apex), and leaves, but canpromote transcription weakly or not at all in tissues such asreproductive tissues of flowers and developing seeds. Non-limitingexamples of broadly expressing promoters that can be included in thenucleic acid constructs provided herein include the p326 (SEQ ID NO:76), YP0144 (SEQ ID NO: 55), YP0190 (SEQ ID NO: 59), p13879 (SEQ ID NO:75), YP0050 (SEQ ID NO: 35), p32449 (SEQ ID NO: 77), 21876 (SEQ ID NO:1), YP0158 (SEQ ID NO: 57), YP0214 (SEQ ID NO: 61), YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO: 26), and PT0633 (SEQ ID NO: 7). Additionalexamples include the cauliflower mosaic virus (CaMV) 35S promoter, themannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV 35S promoter is excluded from the category of broadly expressingpromoters.

Root-active promoters drive transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., drive transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128 (SEQ ID NO: 52), YP0275(SEQ ID NO: 63), PT0625 (SEQ ID NO: 6), PT0660 (SEQ ID NO: 9), PT0683(SEQ ID NO: 14), and PT0758 (SEQ ID NO: 22). Other root-preferentialpromoters include the PT0613 (SEQ ID NO: 5), PT0672 (SEQ ID NO: 11),PT0688 (SEQ ID NO: 15), and PT0837 (SEQ ID NO: 24), which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al. (1989)Proc. Natl. Acad. Sci. USA 86:7890-7894), root cell specific promotersreported by Conkling et al. (1990) Plant Physiol. 93:1203-1211 and thetobacco RD2 gene promoter.

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin gene promoter (Bustos et al. (1989) Plant Cell1(9):839-853), the soybean trypsin inhibitor promoter (Riggs et al.(1989) Plant Cell 1(6):609-621), the ACP promoter (Baerson et al. (1993)Plant Mol Biol, 22(2):255-267), the stearoyl-ACP desaturase gene(Slocombe et al. (1994) Plant Physiol 104(4):167-176), the soybean α′subunit of β-conglycinin promoter (Chen et al. (1986) Proc Natl Acad SciUSA 83:8560-8564), the oleosin promoter (Hong et al. (1997) Plant MolBiol 34(3):549-555), and zein promoters, such as the 15 kD zeinpromoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zeinpromoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoterfrom the rice glutelin-1 gene (Zheng et al. (1993) Mol. Cell Biol.13:5829-5842), the beta-amylase gene promoter, and the barley hordeingene promoter. Other maturing endosperm promoters include the YP0092(SEQ ID NO: 38), PT0676 (SEQ ID NO: 12), and PT0708 (SEQ ID NO: 17).

Promoters that drive transcription in ovary tissues such as the ovulewall and mesocarp can also be useful, e.g., a polygalacturonidasepromoter, the banana TRX promoter, and the melon actin promoter. Othersuch promoters that drive gene expression preferentially in ovules areYP0007 (SEQ ID NO: 30), YP0111 (SEQ ID NO: 46), YP0092 (SEQ ID NO: 38),YP0103 (SEQ ID NO: 43), YP0028 (SEQ ID NO: 33), YP0121 (SEQ ID NO: 51),YP0008 (SEQ ID NO: 31), YP0039 (SEQ ID NO: 34), YP0115 (SEQ ID NO: 47),YP0119 (SEQ ID NO: 49), YP0120 (SEQ ID NO: 50) and YP0374 (SEQ ID NO:68).

In some other embodiments of the present invention, embryo sac/earlyendosperm promoters can be used in order drive transcription of thesequence of interest in polar nuclei and/or the central cell, or inprecursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994)Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039 (SEQ ID NO: 34), YP0101 (SEQ ID NO: 41),YP0102 (SEQ ID NO: 42), YP0110 (SEQ ID NO: 45), YP0117 (SEQ ID NO: 48),YP0119 (SEQ ID NO: 49), YP0137 (SEQ ID NO: 53), DME, YP0285 (SEQ ID NO:64), and YP0212 (SEQ ID NO: 60). Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

Promoters that preferentially drive transcription in zygotic cellsfollowing fertilization can provide embryo-preferential expression andmay be useful for the present invention. Most suitable are promotersthat preferentially drive transcription in early stage embryos prior tothe heart stage, but expression in late stage and maturing embryos isalso suitable. Embryo-preferential promoters include the barley lipidtransfer protein (Ltp1) promoter (Plant Cell Rep (2001) 20:647-654,YP0097 (SEQ ID NO: 40), YP0107 (SEQ ID NO: 44), YP0088 (SEQ ID NO: 37),YP0143 (SEQ ID NO: 54), YP0156 (SEQ ID NO: 56), PT0650 (SEQ ID NO: 8),PT0695 (SEQ ID NO: 16), PT0723 (SEQ ID NO: 19), PT0838 (SEQ ID NO: 25),PT0879 (SEQ ID NO: 28) and PT0740 (SEQ ID NO: 20).

Promoters active in photosynthetic tissue in order to drivetranscription in green tissues such as leaves and stems are ofparticular interest for the present invention. Most suitable arepromoters that drive expression only or predominantly such tissues.Examples of such promoters include the ribulose-1,5-bisphosphatecarboxylase (RbcS) promoters such as the RbcS promoter from easternlarch (Larix laricina), the pine cab6 promoter (Yamamoto et al. (1994)Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat(Fejes et al. (1990) Plant Mol. Biol. 15:921-932), the CAB-1 promoterfrom spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006),the cab1R promoter from rice (Luan et al. (1992) Plant Cell 4:971-981),the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuokaet al. (1993) Proc Natl Acad. Sci USA 90:9586-9590), the tobacco Lhcb1*2promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), theArabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al.(1995) Planta 196:564-570), and thylakoid membrane protein promotersfrom spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Otherpromoters that drive transcription in stems, leafs and green tissue arePT0535 (SEQ ID NO: 3), PT0668 (SEQ ID NO: 2), PT0886 (SEQ ID NO: 29),PR0924 (SEQ ID NO: 265), YP0144 (SEQ ID NO: 55), YP0380 (SEQ ID NO: 70)and PT0585 (SEQ ID NO: 4).

In some other embodiments of the present invention, inducible promotersmay be desired. Inducible promoters drive transcription in response toexternal stimuli such as chemical agents or environmental stimuli. Forexample, inducible promoters can confer transcription in response tohormones such as giberellic acid or ethylene, or in response to light ordrought. Examples of drought inducible promoters are YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO: 26), YP0381 (SEQ ID NO: 71), YP0337 (SEQ ID NO:66), PT0633 (SEQ ID NO: 7), YP0374 (SEQ ID NO: 68), PT0710 (SEQ ID NO:18), YP0356 (SEQ ID NO: 67), YP0385 (SEQ ID NO: 73), YP0396 (SEQ ID NO:74), YP0384 (SEQ ID NO: 72), PT0688 (SEQ ID NO: 15), YP0286 (SEQ ID NO:65), YP0377 (SEQ ID NO: 69), and PD1367 (SEQ ID NO: 78). Examples ofpromoters induced by nitrogen are PT0863 (SEQ ID NO: 27), PT0829 (SEQ IDNO: 23), PT0665 (SEQ ID NO: 10) and PT0886 (SEQ ID NO: 29). An exampleof a promoter induced by salt is rd29A (Kasuga et al. (1999) NatureBiotech 17: 287-291).

Other Promoters: Other classes of promoters include, but are not limitedto, leaf-preferential, stem/shoot-preferential, callus-preferential,guard cell-preferential, such as PT0678 (SEQ ID NO: 13), andsenescence-preferential promoters. Promoters designated YP0086 (SEQ IDNO: 36), YP0188 (SEQ ID NO: 58), YP0263 (SEQ ID NO: 62), PT0758 (SEQ IDNO: 22), PT0743 (SEQ ID NO: 21), PT0829 (SEQ ID NO: 23), YP0119 (SEQ IDNO: 49), and YP0096 (SEQ ID NO: 39), as described in theabove-referenced patent applications, may also be useful.

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component consists of a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component consists of a transgenic plant that comprise anucleic acid molecule of the invention operatively linked to thetarget-binding sequence/region of the transcriptional activator. The twotransgenic plants are crossed and the nucleic acid molecule of theinvention is expressed in the progeny of the plant. In anotheralternative embodiment of the present invention, the misexpression canbe accomplished by having the sequences of the two component systemtransformed in one transgenic plant line.

Another alternative consists in inhibiting expression of a biomass orvigor-modulating polypeptide in a plant species of interest under salineconditions. The term “expression” refers to the process of convertinggenetic information encoded in a polynucleotide into RNA throughtranscription of the polynucleotide (i.e., via the enzymatic action ofan RNA polymerase), and into protein through translation of mRNA.“Up-regulation” or “activation” refers to regulation that increases theproduction of expression products relative to basal or native states,while “down-regulation” or “repression” refers to regulation thatdecreases production relative to basal or native states.

A number of nucleic-acid based methods, including anti-sense RNA,ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be usedto inhibit protein expression in plants. Antisense technology is onewell-known method. In this method, a nucleic acid segment from theendogenous gene is cloned and operably linked to a promoter so that theantisense strand of RNA is transcribed. The recombinant vector is thentransformed into plants, as described above, and the antisense strand ofRNA is produced. The nucleic acid segment need not be the entiresequence of the endogenous gene to be repressed, but typically will besubstantially identical to at least a portion of the endogenous gene tobe repressed. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Typically, a sequence of at least 30nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500nucleotides or more).

Thus, for example, an isolated nucleic acid provided herein can be anantisense nucleic acid to one of the aforementioned nucleic acidsencoding a polypeptide modulates biomass under saline conditions. Anucleic acid that decreases the level of a transcription or translationproduct of a gene encoding a biomass-modulating polypeptide istranscribed into an antisense nucleic acid similar or identical to thesense coding sequence of the biomass- or growth rate-modulatingpolypeptide. Alternatively, the transcription product of an isolatednucleic acid can be similar or identical to the sense coding sequence ofa biomass growth rate-modulating polypeptide, but is an RNA that isunpolyadenylated, lacks a 5′ cap structure, or contains an unsplicableintron.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. (See, U.S. Pat. No.6,423,885). Ribozymes can be designed to specifically pair withvirtually any target RNA and cleave the phosphodiester backbone at aspecific location, thereby functionally inactivating the target RNA.Heterologous nucleic acids can encode ribozymes designed to cleaveparticular mRNA transcripts, thus preventing expression of apolypeptide. Hammerhead ribozymes are useful for destroying particularmRNAs, although various ribozymes that cleave mRNA at site-specificrecognition sequences can be used. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target RNAcontains a 5′-UG -3′ nucleotide sequence. The construction andproduction of hammerhead ribozymes is known in the art. See, forexample, U.S. Pat. No. 5,254,678 and WO 02/46449 and references citedtherein. Hammerhead ribozyme sequences can be embedded in a stable RNAsuch as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.Perriman, et al. (1995) Proc. Natl. Acad. Sci. USA, 92(13):6175-6179; deFeyter and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter 43,“Expressing Ribozymes in Plants”, Edited by Turner, P. C, Humana PressInc., Totowa, N.J. RNA endoribonucleases such as the one that occursnaturally in Tetrahymena thermophila, and which have been describedextensively by Cech and collaborators can be useful. See, for example,U.S. Pat. No. 4,987,071.

Methods based on RNA interference (RNAi) can be used. RNA interferenceis a cellular mechanism to regulate the expression of genes and thereplication of viruses. This mechanism is thought to be mediated bydouble-stranded small interfering RNA molecules. A cell responds to sucha double-stranded RNA by destroying endogenous mRNA having the samesequence as the double-stranded RNA. Methods for designing and preparinginterfering RNAs are known to those of skill in the art; see, e.g., WO99/32619 and WO 01/75164. For example, a construct can be prepared thatincludes a sequence that is transcribed into an interfering RNA. Such anRNA can be one that can anneal to itself, e.g., a double stranded RNAhaving a stem-loop structure. One strand of the stem portion of a doublestranded RNA comprises a sequence that is similar or identical to thesense coding sequence of the polypeptide of interest, and that is fromabout 10 nucleotides to about 2,500 nucleotides in length. The length ofthe sequence that is similar or identical to the sense coding sequencecan be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25nucleotides to 100 nucleotides. The other strand of the stem portion ofa double stranded RNA comprises an antisense sequence of thebiomass-modulating polypeptide of interest, and can have a length thatis shorter, the same as, or longer than the corresponding length of thesense sequence. The loop portion of a double stranded RNA can be from 10nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000nucleotides, from 20 nucleotides to 500 nucleotides, or from 25nucleotides to 200 nucleotides. The loop portion of the RNA can includean intron. See, e.g., WO 99/53050.

Transcriptional silencing of the target gene can also be achieved viathe promoter through expression of an RNAi construct. This results inthe synthesis of double stranded RNA molecules of which the nucleotidessequence is identical to a part of the promoter region of the targetgene.

Another alternative method for suppression of the target gene may beachieved through a methodology generally referred to as Virus InducedGene Silencing or VIGS (Ratcliff et al (2001) Plant J. 25, 237-245).Here, effective and specific gene silencing is achieved by infection ofa plant with a plant virus carrying an insert which is homologous to thetarget gene. The advantage of the VIGS system is that there is no needto develop a plant transformation protocol for the plant species inwhich the target gene resides.

In all of these silencing methods, the silencing construct (antisenseRNA, co-suppression, RNAi or hairpin construct or VIGs vector)preferably contains a DNA fragment that is identical to the targetsequence (gene or promoter) that needs to be silenced. The percentage ofidentity may, however, range between 50-100%, preferably between60-100%, more preferably between 70-100%, even more preferably between80-100% and most preferably between 90-100%.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev.,7:187-195; Hyrup et al., 1996, Bioorgan. Med. Chem., 4: 5-23. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

Transformation

Nucleic acid molecules of the present invention may be introduced intothe genome or the cell of the appropriate host plant by a variety oftechniques. These techniques, able to transform a wide variety of higherplant species, are well known and described in the technical andscientific literature (see, e.g., Weising et al. (1988) Ann. Rev.Genet., 22:421 and Christou (1995) Euphytica, 85:13-27).

A variety of techniques known in the art are available for theintroduction of DNA into a plant host cell. These techniques includetransformation of plant cells by injection (Newell (2000) Mol Biotech16:53-65), microinjection (Griesbach (1987) Plant Sci. 50:69-77),electroporation of DNA (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA82:5824), PEG (Paszkowski et al. (1984) EMBO J. 3:2717), use ofbiolistics (Klein et al. (1987) Nature 327:773), fusion of cells orprotoplasts (Willmitzer, L. (1993) Transgenic Plants. In: Iotechnology,A Multi-Volume Comprehensive treatise (H. J. Rehm, G. Reed, A. Püler, P.Stadler, eds., Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge),and via T-DNA using Agrobacterium tumefaciens (Crit. Rev. Plant. Sci.4:1-46; Fromm et al. (1990) Biotechnology 8:833-844) or Agrobacteriumrhizogenes (Cho et al. (2000) Planta 210:195-204) or other bacterialhosts (Brootghaerts et al. (2005) Nature 433:629-633), for example.

In addition, a number of non-stable transformation methods that are wellknown to those skilled in the art may be desirable for the presentinvention. Such methods include, but are not limited to, transientexpression (Lincoln et al. (1998) Plant Mol. Biol. Rep. 16:1-4) andviral transfection (Lacomme et al. (2001), “Genetically EngineeredViruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOSScientific Publishers, Ltd. Oxford, UK).

Seeds are obtained from the transformed plants and used for testingstability and inheritance. Generally, two or more generations arecultivated to ensure that the phenotypic feature is stably maintainedand transmitted.

A person of ordinary skill in the art recognizes that after theexpression cassette is stably incorporated in transgenic plants andconfirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

In aspects related to making transgenic plants, a typical step involvesselection or screening of transformed plants, e.g., for the presence ofa functional vector as evidenced by expression of a selectable marker.Selection or screening can be carried out among a population ofrecipient cells to identify transformants using selectable marker genessuch as herbicide resistance genes. Physical and biochemical methods canbe used to identify transformants. These include Southern analysis orPCR amplification for detection of a polynucleotide; Northern blots, S1RNase protection, primer-extension, or RT-PCR amplification fordetecting RNA transcripts; enzymatic assays for detecting enzyme orribozyme activity of polypeptides and polynucleotides; and protein gelelectrophoresis, Western blots, immunoprecipitation, and enzyme-linkedimmunoassays to detect polypeptides. Other techniques such as in situhybridization, enzyme staining, and immunostaining also can be used todetect the presence or expression of polypeptides and/orpolynucleotides. Methods for performing all of the referenced techniquesare known.

A population of transgenic plants can be screened and/or selected forthose members of the population that have a desired trait or phenotypeconferred by expression of the transgene. For example, a population ofprogeny of a single transformation event can be screened for thoseplants having a desired level of expression of a heterologous salttolerance polypeptide or nucleic acid. As an alternative, a populationof plants comprising independent transformation events can be screenedfor those plants having a desired trait, such as salt tolerance.Selection and/or screening can be carried out over one or moregenerations, which can be useful to identify those plants that have astatistically significant difference in a protein level as compared to acorresponding level in a control plant. Selection and/or screening canalso be carried out in more than one geographic location. In some cases,transgenic plants can be grown and selected under conditions whichinduce a desired phenotype or are otherwise necessary to produce adesired phenotype in a transgenic plant. In addition, selection and/orscreening can be carried out during a particular developmental stage inwhich the phenotype is expected to be exhibited by the plant. Selectionand/or screening can be carried out to choose those transgenic plantshaving a statistically significant difference in salt tolerance relativeto a control plant that lacks the transgene. Selected or screenedtransgenic plants have an altered phenotype as compared to acorresponding control plant, as described elsewhere in thisspecification.

The nucleic acid molecules of the present invention may be used toconfer the trait of improved tolerance to saline conditions. Theinvention has utility in improving important agronomic characteristicsof crop plants, for example enabling plants to be productivelycultivated in saline conditions. As noted above, transgenic plants thatexhibit overexpression of the polynucleotides of the invention grow wellunder high salt conditions.

The nucleic acid molecules of the present invention encode appropriateproteins from any organism, but are preferably found in plants, fungi,bacteria or animals.

Transgenic Plant Phenotypes

Information that the polypeptides disclosed herein can modulate salttolerance is useful in breeding of crop plants. Based on the effect ofthe disclosed polypeptides on salt tolerance, one can search for andidentify polymorphisms linked to genetic loci for such polypeptides.Polymorphisms that can be identified include simple sequence repeats(SSRs), amplified fragment length polymorphisms (AFLPs) and restrictionfragment length polymorphisms (RFLPs).

If a polymorphism is identified, its presence and frequency inpopulations is analyzed to determine if it is statisticallysignificantly correlated to an increase in salt tolerance. Thosepolymorphisms that are correlated with an increase in salt tolerance canbe incorporated into a marker assisted breeding program to facilitatethe development of lines that have a desired increase in salt tolerance.Typically, a polymorphism identified in such a manner is used withpolymorphisms at other loci that are also correlated with a desiredincrease in salt tolerance or other desired trait.

The methods according to the present invention can be applied to anyplant, preferably higher plants, pertaining to the classes ofAngiospermae and Gymnospermae. Plants of the subclasses of theDicotylodenae and the Monocotyledonae are particularly suitable.Dicotyledonous plants belonging to the orders of the Magniolales,Illiciales, Laurales, Piperales Aristochiales, Nymphaeales,Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales,Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales,Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales,Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales,Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales,Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales,Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales, for example, are alsosuitable. Monocotyledonous plants belonging to the orders of theAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Lilliales, and Orchidales also may be useful in embodiments of thepresent invention. Further examples include, but are not limited to,plants belonging to the class of the Gymnospermae are Pinales,Ginkgoales, Cycadales and Gnetales.

The methods of the present invention are preferably used in plants thatare important or interesting for agriculture, horticulture, biomass forbioconversion and/or forestry. Non-limiting examples include, forinstance, tobacco, oilseed rape, sugar beet, potatoes, tomatoes,cucumbers, peppers, beans, peas, citrus fruits, avocados, peaches,apples, pears, berries, plumbs, melons, eggplants, cotton, soybean,sunflowers, roses, poinsettia, petunia, guayule, cabbages, spinach,alfalfa, artichokes, sugarcane, mimosa, Servicea lespedera, corn, wheat,rice, rye, barley, sorghum and grasses such as switch grass, giant reed,Bermuda grass, Johnson grasses or turf grass, millet, hemp, bananas,poplars, eucalyptus trees and conifers. Of interest are plants grown forenergy production, so called energy crops, such as broadleaf plants likealfalfa, hemp, Jerusalem artichoke and grasses such as sorghum,switchgrass, Johnson grass and the likes. Thus, the described materialsand methods are useful for modifying biomass characteristics, such ascharacteristics of biomass renewable energy source plants. A biomassrenewable energy source plant is a plant having or producing material(either raw or processed) that comprises stored solar energy that can beconverted to fuel. In general terms, such plants comprise dedicatedenergy crops as well as agricultural and woody plants. Examples ofbiomass renewable energy source plants include: switchgrass, elephantgrass, giant chinese silver grass, energycane, giant reed (also known aswild cane), tall fescue, bermuda grass, sorghum, napier grass, alsoknown as uganda grass, triticale, rye, winter wheat, shrub poplar, shrubwillow, big bluestem, reed canary grass and corn.

Homologues Encompassed by the Invention

It is known in the art that one or more amino acids in a sequence can besubstituted with other amino acid(s), the charge and polarity of whichare similar to that of the substituted amino acid, i.e. a conservativeamino acid substitution, resulting in a biologically/functionally silentchange. Conservative substitutes for an amino acid within thepolypeptide sequence can be selected from other members of the class towhich the amino acid belongs. Amino acids can be divided into thefollowing four groups: (1) acidic (negatively charged) amino acids, suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as serine, threonine, tyrosine, asparagine, and glutamine;and (4) neutral nonpolar (hydrophobic) amino acids such as glycine,alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, cysteine, and methionine.

Nucleic acid molecules of the present invention can comprise sequencesthat differ from those encoding a protein or fragment thereof selectedfrom the group consisting of SEQ ID NOs. 80, 99, 106, 123, 132, 146, 154and 172, respectively, due to the fact that the different nucleic acidsequence encodes a protein having one or more conservative amino acidchanges.

Biologically functional equivalents of the polypeptides, or fragmentsthereof, of the present invention can have about 10 or fewerconservative amino acid changes, more preferably about 7 or fewerconservative amino acid changes, and most preferably about 5 or fewerconservative amino acid changes. In a preferred embodiment of thepresent invention, the polypeptide has between about 5 and about 500conservative changes, more preferably between about 10 and about 300conservative changes, even more preferably between about 25 and about150 conservative changes, and most preferably between about 5 and about25 conservative changes or between 1 and about 5 conservative changes.

Identification of Useful Nucleic Acid Molecules and Their CorrespondingNucleotide Sequences

The nucleic acid molecules, and nucleotide sequences thereof, of thepresent invention were identified by use of a variety of screens thatare predictive of nucleotide sequences that provide plants with improvedvegetative growth, growth rate, and/or biomass under saline conditions.One or more of the following screens were, therefore, utilized toidentify the nucleotide (and amino acid) sequences of the presentinvention.

The present invention is further exemplified by the following examples.The examples are not intended to in any way limit the scope of thepresent application and its uses.

6. Experiments Confirming the Usefulness of the Polynucleotides andPolypeptides of the Invention General Protocols Agrobacterium-MediatedTransformation of Arabidopsis

Host Plants and Transgenes: Wild-type Arabidopsis thaliana Wassilewskija(WS) plants are transformed with Ti plasmids containing nucleic acidsequences to be expressed, as noted in the respective examples, in thesense orientation relative to the 35S promoter in a Ti plasmid. A Tiplasmid vector useful for these constructs, CRS 338, contains theCeres-constructed, plant selectable marker gene phosphinothricinacetyltransferase (PAT), which confers herbicide resistance totransformed plants.

Ten independently transformed events are typically selected andevaluated for their qualitative phenotype in the T₁ generation.

Preparation of Soil Mixture: 24L Sunshine Mix #5 soil (Sun GroHorticulture, Ltd., Bellevue, Wash.) is mixed with 16L Therm-O-Rockvermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixerto make a 60:40 soil mixture. To the soil mixture is added 2 TbspMarathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE®14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer20-20-20 (J.R. Peters, Inc., Allentown, Pa.), which are first added to 3gallons of water and then added to the soil and mixed thoroughly.Generally, 4-inch diameter pots are filled with soil mixture. Pots arethen covered with 8-inch squares of nylon netting.

Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated.25 drops are added to each pot. Clear propagation domes are placed ontop of the pots that are then placed under 55% shade cloth andsubirrigated by adding 1 inch of water.

Plant Maintenance: 3 to 4 days after planting, lids and shade cloth areremoved. Plants are watered as needed. After 7-10 days, pots are thinnedto 20 plants per pot using forceps. After 2 weeks, all plants aresubirrigated with Peters fertilizer at a rate of 1 Tsp per gallon ofwater. When bolts are about 5-10 cm long, they are clipped between thefirst node and the base of stem to induce secondary bolts. Dippinginfiltration is performed 6 to 7 days after clipping.

Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL eachof carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stockconcentration). Agrobacterium starter blocks are obtained (96-well blockwith Agrobacterium cultures grown to an OD₆₀₀ of approximately 1.0) andinoculated one culture vessel per construct by transferring 1 mL fromappropriate well in the starter block. Cultures are then incubated withshaking at 27° C. Cultures are spun down after attaining an OD₆₀₀ ofapproximately 1.0 (about 24 hours). 200 mL infiltration media is addedto resuspend Agrobacterium pellets. Infiltration media is prepared byadding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/ml benzylaminopurineto 900 ml water.

Dipping Infiltration: The pots are inverted and submerged for 5 minutesso that the aerial portion of the plant is in the Agrobacteriumsuspension. Plants are allowed to grow normally and seed is collected.

High-throughput Phenotypic Screening of Misexpression Mutants: Seed isevenly dispersed into water-saturated soil in pots and placed into adark 4° C. cooler for two nights to promote uniform germination. Potsare then removed from the cooler and covered with 55% shade cloth for4-5 days. Cotyledons are fully expanded at this stage. FINALE® (SanofiAventis, Paris, France) is sprayed on plants (3 ml FINALE® diluted into48 oz. water) and repeated every 3-4 days until only transformantsremain.

Screening: Screening is routinely performed by high-salt agar plateassay and also by high-salt soil assay. Traits assessed in high-saltconditions include: seedling area, photosynthesis efficiency, saltgrowth index, and regeneration ability.

-   -   Seedling area: the total leaf area of a young plant about 2        weeks old.    -   Photosynthesis efficiency (Fv/Fm): Seedling photosynthetic        efficiency, or electron transport via photosystem II, is        estimated by the relationship between Fm, the maximum        fluorescence signal and the variable fluorescence, Fv. Here, a        reduction in the optimum quantum yield (Fv/Fm) indicates stress,        and so can be used to monitor the performance of transgenic        plants compared to non-transgenic plants under salt stress        conditions.    -   Salt growth index=seedling area×photosynthesis efficiency        (Fv/Fm).

PCR was used to amplify the DNA insert in one randomly chosen T₂ plant.This PCR product was then sequenced to confirm the sequence in theplants.

Assessing Tolerance to Salt Stress: Initially, independently transformedplant lines are selected and qualitatively evaluated for their toleranceto salt stress in the T₁ generation. The transformed lines thatqualitatively show the strongest tolerance to salt stress in the T₁generation are selected for further evaluation in the T₂ and T₃generations. This evaluation involves sowing seeds from the selectedtransformed plant lines on MS agar plates containing either 100 mM or150 mM NaCl and incubating the seeds for 5 to 14 days to allow forgermination and growth.

Calculating SGI: After germination and growth, seedling area andphotosynthesis efficiency of transformed lines and a wild-type controlare determined. From these measurements, the Salt Growth Index (SGI) iscalculated and compared between wild-type and transformed seedlings. TheSGI calculation is made by averaging seedling area and photosynthesisefficiency measurements taken from two replicates of 36 seedlings foreach transformed line and a wild-type control and performing a t-test.

Determining Transgene Copy Number: T₂ generation transformed plants aretested on BASTA™ plates in order to determine the transgene copy numberof each transformed line. A BASTA™ resistant:BASTA™ sensitivesegregation ratio of 3:1 generally indicates one copy of the transgene.

Results:

The following Examples provide information for polynucleotides and theirencoded polypeptides useful for increasing tolerance to salt stress.Enhanced salt tolerance gives the opportunity to grow crops in salineconditions without stunted growth and diminished yields due tosalt-induced ion imbalance, disruption of water homeostasis, inhibitionof metabolism, damage to membranes, and/or cell death. The ability togrow crops in saline conditions would result in an overall expansion ofarable land and increased output of land currently marginally productivedue to elevated salinity

EXAMPLE 1 ME03807 (Ceres Clone 8686; SEQ ID No. 79)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone8686. Two transformed lines, ME03807-02 and ME03807-03, showed thestrongest qualitative tolerance to salt stress in a prevalidation assay(Table 1-1). Their tolerance to 150 mM NaCl was further evaluated in avalidation assay for two generations. Segregation ratios (BASTA™resistant: BASTA™ sensitive) indicated that ME03807-02 contains twocopies of the transgene and that ME03807-03 carries one copy of thetransgene.

TABLE 1-1 Prevalidation assay of ME03807 salt tolerance as compared towild-type Ws WS Wild-type ME03807-02 ME03807-03 Mean* 0.0268 0.03970.0506 Standard Error 0.0006 0.0041 0.0038 *Average seedling area of 36plants grown on MS agar plates containing 150 mM NaCl for 14 days

When grown on MS agar plates containing 150 mM NaCl, ME03807-02 andME03807-03 transgenic plants showed significantly increased seedlingarea and SGI relative to non-transgenic plants. As shown in Table 1-2,the T2-generation SGI value for ME03807-02 seedlings increased by 74.4%while ME03807-03 seedlings increased by 87.6% compared to non-transgeniccontrol seedlings. In the T₃ generation, the SGI increase was 134.2% forME03807-02 and 141.8% for ME03807-03. The differences between transgenicand non-transgenic seedlings are statistically significant under thet-test, and clearly demonstrate that the enhanced tolerance to saltstress is a result of the ectopic expression of Ceres Clone 8686 in theME03807 transformant lines.

TABLE 1-2 Validation assay of ME03807 salt stress tolerance in twogenerations SGI of SGI* of pooled non- t-Test % of ME transgenicstransgenics t- SGI Events Avg SE N Avg SE N value t_(0.05) increaseME03807- 0.952 0.126 32 0.546 0.087 24 2.66 1.68 74.4 02T₂ ME03807-0.604 0.047 24 0.322 0.065 13 3.51 1.70 87.6 03T₂ ME03807- 0.965 0.11119 0.412 0.031 11 5.95 1.70 134.2 02T₃ ME03807- 1.064 0.104 42 0.4400.028 16 9.60 1.68 141.8 03T₃ *SGI (Salt Growth Index) = seedling area ×Fv/Fm (photosynthesis efficiency)

Summary of Results:

-   -   Ectopic expression of Ceres Clone 8686 under the control of the        35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type WS seedlings.    -   The protein encoded by Ceres Clone 8686 is a 255-amino-acid        putative cyclase (Susstrunk et al. (1998) Mol Microbiol,        30(1):33-46 and Kang et al. (1999) Microbiology, 145:1161-72.        Cyclase is a large gene family that includes adenylyl cyclase,        which converts ATP to cAMP. cAMP is an important signal molecule        that is involved in signal transduction which conveys signals        from a plasma membrane receptor to cytosol cascades. The ME03807        transgene is more closely related to cyclase enzymes that are        involved in antibiotic synthesis.

EXAMPLE 2 ME00774 (Ceres Clone 2767; SEQ ID No. 131)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 32449 promoter (SEQ ID No. 77) and Ceres Clone2767. Two transformed lines, ME00774-03 and ME00774-04, showed thestrongest qualitative tolerance to salt stress in a prevalidation assay(Table 2-1). Their tolerance to 150 mM NaCl was further evaluated in avalidation assay for two generations. Segregation ratios (BASTA™resistant:BASTA™ sensitive) indicated that ME00774-03 contains twocopies of the transgene and that ME00774-04 carries one copy of thetransgene.

TABLE 2-1 Prevalidation assay of ME00774 salt tolerance as compared towild-type Ws Ws wild- ME00774- ME00774- ME00774- ME00774- ME00774- type01 02 03 04 05 Mean* 0.0286 0.0313 0.0333 0.0468 0.0384 0.0343 Std0.0006 0.0015 0.0019 0.0037 0.0026 0.0024 Error *Average seedling areaof 36 plants grown on MS agar plates containing 150 mM NaCl for 14 days

When grown on MS agar plates containing 150mM NaCl, ME00774-03 andME00774-04 transgenic plants showed significantly greater seedling areaand SGI relative to non-transgenic plants. As shown in Table 2-2, theT2-generation SGI value for ME00774-03 seedlings increased by 41.8%while ME00774-04 seedlings increased by 379.4% compared tonon-transgenic control seedlings. In the T₃ generation, the SGI increasewas 315.1% for ME00774-03 and 551.8% for ME00774-04. The differencesbetween transgenic and non-transgenic seedlings are statisticallysignificant under the t-test, and clearly demonstrate that the enhancedtolerance to salt stress was a result of the ectopic expression of CeresClone 2767 in the ME00774 transformant lines.

TABLE 2-2 Validation assay of ME00774 on salt tolerance in twogenerations SGI* of SGI of pooled t-Test % of ME transgenicsnon-transgenics t- SGI Events Avg SE N Avg SE N value t_(0.05) increaseME00774- 1.462 0.122 51 1.031 0.127 18 2.45 1.67 41.8 03T₂ ME00774-0.954 0.072 20 0.199 0.03 10 9.78 1.70 379.4 04T₂ ME00774- 1.598 0.08148 0.385 0.05 23 12.79 1.67 315.1 03T₃ ME00774- 1.082 0.091 20 0.1660.032 16 9.52 1.70 551.8 04T₃ *SGI (Salt Growth Index) = seedling area ×Fv/Fm (photosynthesis efficiency)

Summary of Results:

-   -   Ectopic expression of Clone 2767 under the control of the 32449        promoter enhances tolerance to salt stress.    -   Ceres Clone 2767 encodes a 154-amino-acid protein that belongs        to a universal stress protein family (Kerk et al. (2003) Plant        Physiol. 131 (3) : 1209-19). The USP superfamily has its members        conserved in bacteria, archaea, and eukaryotes. The expression        of USP genes in E. coli is induced by a large variety of        environmental insults. The uspA gene plays an important role        for E. coli to survive in cellular growth arrest, but the        molecular mechanism of the gene function is not known yet        (Nachin et al. (2005) J Bacteriol 187(18):6265-72). In        Arabidopsis, there are 44 family members of USP. However, their        function has not been characterized yet (Kerk et al. 2003). A        rice homolog, OsUsp1, has been found to be induced by        submergence and ethylene (Sauter et al. (2002) J Exp Bot        53(379):2325-31).    -   The identification of an AtUsp gene in a salt screen suggests        that the Arabidopsis USP family members may play a similar role        in stress tolerance as observed in E. coli.

EXAMPLE 3 ME0146 (Ceres Clone 16403; SEQ ID No. 145)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 16403. Two transformedlines, ME01468-01 and ME01468-04, showed the strongest qualitativetolerance to salt stress in a prevalidation assay (Table 3-1). Theirtolerance to 100 mM NaCl was further evaluated in a validation assay fortwo generations. Segregation ratios (BASTA™ resistant: BASTA™ sensitive)indicated ME01468-01 and ME01468-04 transformed lines each carry onecopy of the transgene.

TABLE 3-1 Prevalidation assay of ME00774 salt tolerance as compared towild-type Ws Ws Wild- ME01468- ME01468- ME01468- ME01468- type 01 02 0304 Mean* 0.0268 0.0424 0.0312 0.0215 0.0395 Standard 0.0006 0.00320.0018 0.0027 0.0031 Error *Average seedling area of 36 plants grown onMS agar plates containing 150 mM NaCl for 14 days

When grown on MS agar plates containing 100 mM NaCl, ME01468-01 andME01468-04 transgenic plants showed significantly greater seedling areaand SGI relative to non-transgenic plants. As shown in Table 3-1, theT2-generation SGI value for ME01468-01 seedlings increased by 23.7%while ME01468-04 seedlings increased by 39.3% compared to non-transgeniccontrol seedlings. In the T₃ generation, the SGI increase was 83.7% forME01468-01 and 79.4% for ME01468-04. The differences between transgenicand non-transgenic seedlings are statistically significant under thet-test, and clearly demonstrate that the enhanced tolerance to saltstress was a result of the ectopic expression of Ceres Clone 16403 inthe ME01468 transformant lines.

TABLE 3-2 Validation assay of ME01468 on salt tolerance in twogenerations SGI* of SGI of pooled t-Test % of ME transgenicsnon-transgenics t- SGI Events Avg SE N Avg SE N value t_(0.05) increaseME01468- 3.842 0.146 39 3.105 0.307 29 2.162 1.67 23.7 01T₂ ME01468-3.143 0.179 35 2.256 0.261 32 2.795 1.67 39.3 04T₂ ME01468- 5.939 0.41633 3.233 0.296 37 5.293 1.67 83.7 01T₃ ME01468- 7.508 0.524 13 4.1860.469 21 4.719 1.70 79.4 04T₃ *SGI (Salt Growth Index) = seedling area ×Fv/Fm (photosynthesis efficiency

Summary of Results:

-   -   Ectopic expression of Ceres Clone 16403 under the control of the        35S promoter enhances tolerance to high salt stress.    -   Ceres Clone 16403 encodes a 238-amino-acid calcium-binding        protein that also shows similarity to an oxygen evolving complex        from rice (Sanchez-Barrena et al. (2005) J Mol Biol.        345(5):1253-64). It is worth noting that SOS3, an important gene        involved in salt tolerance, has been molecularly characterized        as a Ca⁺⁺ binding protein.

EXAMPLE 4 ME02064 (Ceres Clone 375578; SEQ ID No.98)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 375578. Threetransformed lines, ME02064-01 and ME02064-03, ME02064-04, showed thestrongest qualitative tolerance to salt stress in a prevalidation assay(Table 4-1). Their tolerance to 150 mM NaCl was further evaluated in avalidation assay for two generations. Segregation ratios (BASTA™resistant: BASTA™ sensitive) indicated ME02064-01 and ME02064-03,ME02064-04 transformed lines each carry one copy of the transgene.

TABLE 4-1 Prevalidation assay of ME02064 salt tolerance as compared towild-type Ws Ws Wild- ME02064- ME02064- ME02064- ME02064- ME02064- type01 02 03 04 05 Mean* 0.0359 0.0435 0.0346 0.0441 0.0438 0.0305 StandardError 0.0016 0.0048 0.004 0.0041 0.0035 0.0019 *Average seedling area of36 plants grown on MS agar plates containing 150 mM NaCl for 14 days

When grown on MS agar plates containing 150 mM NaCl, ME02064-01 andME02064-03, ME02064-04 transgenic plants showed significantly greaterseedling area and SGI relative to non-transgenic plants. As shown inTable 4-2, the T2-generation SGI value for ME02064-01 seedlingsincreased by 110% while ME02064-03 seedlings increased by 131% andME02064-04 seedlings increased by 72% compared to non-transgenic controlseedlings. In the T₃ generation, the SGI increase was 43% forME02064-01, 47% for ME02064-03, and 64% for ME02064-04. The differencesbetween transgenic and non-transgenic seedlings are statisticallysignificant, and clearly demonstrate that the enhanced tolerance to saltstress was a result of the ectopic expression of Ceres Clone 375578 inthe ME02064 transformant lines.

TABLE 4-2 Validation assay of ME02064 on salt tolerance in twogenerations SGI* of SGI of pooled t-Test % of ME transgenicsnon-transgenics t- SGI Events Avg SE N Avg SE N value t_(0.05) increaseME02064- 2.057 0.249 12 0.977 0.205 17 3.35 1.70 110.5 01-T₂ ME02064-2.237 0.371 5 0.968 0.140 24 3.20 1.70 131.1 03-T₂ ME02064- 1.810 0.14614 1.055 0.135 13 3.81 1.70 71.6 04-T₂ ME02064- 2.438 0.170 21 1.7080.289 9 2.18 1.70 42.7 01-T₃ ME02064- 2.837 0.257 20 1.927 0.271 14 2.431.70 47.2 03-T₃ ME02064- 2.770 0.318 16 1.688 0.188 19 2.93 1.70 64.104-T₃ *SGI (Salt Growth Index) = seedling area × Fv/Fm (photosynthesisefficiency)

Summary of Results:

-   -   Ectopic expression of Ceres Clone 375578 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.    -   Ceres Clone 375578 encodes a 311-amino-acid protein that belongs        to the calmodulin binding family (Sanchez-Barrena et al. (2005)        J Mol Biol. 345(5):1253-64). Ca⁺⁺ homeostasis is an important        signaling cascade in abiotic and biotic resistance. A critical        gene, SOS3, involved in salt tolerance has been previously        identified to be a Ca⁺⁺ binding protein. Understanding the        connection between SOS3 and the transgene in ME02064 will help        to better engineer resistance to salt stress.

EXAMPLE 5: ME04074 (Ceres clone 105319; SEQ ID No. 105)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 105319. Twotransformed lines, ME04074-02 and ME04074-05, showed the strongestqualitative tolerance to salt stress in a prevalidation assay (Table5-1). Their tolerance to 150 mM NaCl was further evaluated in avalidation assay for two generations. Segregation ratios (BASTA™resistant: BASTA™ sensitive) indicate ME04074-02 and ME04074-05transformed lines each carry one copy of the transgene.

TABLE 5-1 Prevalidation assay of ME04074 salt tolerance as compared towild-type Ws Ws Wild- ME04074- ME04074- ME04074- ME04074- ME04074- type01 02 03 04 05 Mean* 0.0301 0.0332 0.0423 0.0351 0.039 0.0448 StandardError 0.0032 0.0027 0.0033 0.0026 0.0025 0.0027 *Average seedling areaof 36 plants grown on MS agar plates containing 150 mM NaCl for 14 days

When grown on MS agar plates containing 150 mM NaCl, ME04074-02 andME04074-05 transgenic plants showed significantly greater seedling areaand SGI relative to non-transgenic plants. As shown in Table 5-2, theT2-generation SGI value for ME04074-02 seedlings increased by 40.6%while ME04074-05 seedlings increased by 52.2% compared to non-transgeniccontrol seedlings. In the T₃ generation, the SGI increase was 18.5% forME04074-02 and 60.6% for ME04074-05. The differences between transgenicand non-transgenic seedlings are statistically significant under thet-test, and clearly demonstrate that the enhanced tolerance to saltstress was a result of the ectopic expression of Ceres Clone 105319 inthe ME04074 transformant lines.

TABLE 5-2 Validation assay of ME04074 on salt tolerance in twogenerations SGI* of SGI of pooled t-Test % of ME transgenicsnon-transgenics t- SGI Events Avg SE N Avg SE N value t_(0.05) increaseME04074- 2.432 0.212 23 1.730 0.155 40 2.68 1.67 40.6 0₂-T₂ ME04074-2.707 0.212 26 1.778 0.171 38 3.41 1.67 52.2 0₅-T₂ ME04074- 2.257 0.15634 1.905 0.190 34 1.43 1.67 18.5 0₂-T₃ ME04074- 2.851 0.158 32 1.7750.147 52 4.98 1.67 60.6 0₅-T₃ *SGI (Salt Growth Index) = seedling area ×Fv/Fm (photosynthesis efficiency)

Summary of Results:

-   -   Ectopic expression of Ceres Clone 105319 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.    -   The protein encoded by Ceres Clone 105319 encodes a putative        shikimate cyclase (Griffen et al. (1995) DNA Seq 5(3):195-197).        The enzyme has ATP binding activity and catalyzes the fifth step        in the biosynthesis of aromatic amino acids from chorismate. The        protein is found in bacteria, fungi and plants. How this protein        is involved in stress response is not yet known. However,        aromatic acids, such as L-phenylalanine, are important        substrates for the phenylpropanoid biosynthesis pathway, which        produces many compounds related to stress responses.

EXAMPLE 6 ME02907 (Ceres Clone 29658; SEQ ID No.122)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 29658. Threetransformed lines, ME02907-01, ME02907-03 and ME02907-05, showed thestrongest qualitative tolerance to salt stress in a prevalidation assay(Table 6-1). Their tolerance to 150 mM NaCl was further evaluated in avalidation assay using ME02907-03 and ME02907-05 for two generations.Segregation ratios (BASTA™ resistant: BASTA™ sensitive) indicatedME02907-03 and ME02907-05 transformed lines each carry one copy of thetransgene.

TABLE 6-1 Prevalidation assay of ME02907 salt tolerance as compared towild-type Ws Ws Wild- ME02907- ME02907- ME02907- ME02907- type 01 03 0405 Mean* 0.0268 0.034483 0.0315 0.0224 0.0368 Standard 0.0006 0.0020160.0029 0.0031 0.0039 Error *Average seedling area of 36 plants grown onMS agar plates containing 150 mM NaCl for 14 days

When grown on MS agar plates containing 150mM NaCl, ME02907-03 andME02907-05 transgenic plants showed significantly greater seedling areaand SGI relative to non-transgenic plants. As shown in Table 6-2, theT2-generation SGI value for ME02907-03 seedlings increased by 59% whileME02907-05 seedlings increased by 67% as compared to non-transgeniccontrol seedlings. In the T₃ generation, the SGI increase was 110% forME02907-03 and 99% for ME02907-05. The differences between transgenicand non-transgenic seedlings are statistically significant under thet-test, and clearly demonstrate that the enhanced tolerance to saltstress was a result of the ectopic expression of Ceres Clone 29658 inthe ME02907 transformant lines.

TABLE 6-2 Validation assay of ME02907 on salt tolerance in twogenerations SGI* of SGI of pooled t-Test % of ME transgenicsnon-transgenics t- SGI Events Avg SE N Avg SE N value t_(0.05) increaseME02907- 1.252 0.115 31 0.787 0.121 18 2.79 1.68 59.1 03-T₂ ME02907-1.235 0.120 34 0.738 0.100 28 3.18 1.67 67.3 05-T₂ ME02907- 1.039 0.10026 0.495 0.023 15 7.40 1.69 109.9 03-T₃ ME02907- 1.157 0.064 37 0.5820.070 17 7.53 1.70 98.8 05-T₃ *SGI (Salt Growth Index) = seedling area ×Fv/Fm (photosynthesis efficiency)

Summary of Results:

-   -   Ectopic expression of Ceres Clone 29658 under the control of the        35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.    -   The protein encoded by Ceres Clone 29658 is a putative        calmodulin. Sanchez-Barrena et al. J Mol Biol. 345(5):1253-64.        Ca⁺⁺-mediated signaling is critical in salt tolerance. SOS3 has        been demonstrated to confer salt tolerance in Arabidopsis and it        has Ca⁺⁺ -binding activity.

EXAMPLE 7 ME00199 (Ceres Clone 3964; SEQ ID No. 153)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 32499 promoter and Ceres Clone 3964. Twotransformed lines, ME00199-02 and ME00199-03, showed the strongestqualitative tolerance to salt stress in a prevalidation assay (Table7-1). Their tolerance to 100 mM NaCl was further evaluated in avalidation assay for two generations. Segregation ratios (BASTA™resistant: BASTA™ sensitive) indicated ME00199-02 and ME00199-03transformed lines each carry one copy of the transgene.

TABLE 7-1 Prevalidation assay of ME00199 salt tolerance as compared towild-type Ws Ws wild- ME00199- ME00199- ME00199- type 01-01 02-01 03-01Mean* 0.0268 0.0244 0.0401 0.0307 Standard 0.0006 0.0025 0.0052 0.0037Error *Average seedling area of 36 plants grown on MS agar platescontaining 150 mM NaCl for 14 days

When grown on MS agar plate containing 100 mM NaCl, ME00199-02 andME00199-03 transgenic plants showed significantly greater seedling areaand SGI relative to non-transgenic plants. As shown in Table 7-2, theSGI value of T2-generation ME00199-02 seedlings increased by 106.6% andthe SGI value of T₂-generation ME00199-03 seedlings increased by 48.2%as compared to non-transgenic control seedlings. In the T₃ generation,the SGI increase was 174.3% for ME00199-02 and 205.9% for ME00199-03.The differences between transgenic and non-transgenic seedlings arestatistically significant under the t-test, and clearly demonstrate thatthe enhanced tolerance to salt stress was a result of the ectopicexpression of Ceres Clone 3964 in the ME00199 transformant lines.

TABLE 7-2 Validation assay of ME00199 on salt tolerance in twogenerations SGI* of SGI of pooled % of transgenics non-transgenicst-Test SGI ME Events Avg SE N Avg SE N t-value t_(0.05) increaseME00199- 4.6025 0.3400 43 2.2277 0.2159 28 5.90 1.67 106.6 02T₃ ME00199-3.8795 0.3444 40 2.6182 0.3855 28 2.44 1.67 48.2 03T₃ ME00199- 6.87430.5132 45 2.5058 0.5904 12 5.58 1.68 174.3 02T₄ ME00199- 7.4472 0.739230 2.4343 0.5283 15 5.52 1.68 205.9 03T₄ *SGI (Salt Growth Index) =seedling area × Fv/Fm (photosynthesis efficiency)

Summary of Results:

-   -   Ectopic expression of Ceres Clone 3964 under the control of the        32499 promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.    -   The protein encoded by Ceres Clone 3964 is a putative steroid        sulfotransferase (351 AA).

EXAMPLE 8 ME09814 (Ceres Clone 965405; SEQ ID No. 171)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 965405. Twotransformed lines, ME09814-01 and ME09814-02, showed the strongestqualitative tolerance to salt stress in a prevalidation assay. Theirtolerance to 100 mM NaCl was further evaluated in a validation assay fortwo generations. Segregation ratios (BASTA™ resistant: BASTA™ sensitive)indicated ME09814-01 and ME09814-02 transformed lines each carry onecopy of the transgene originated from Brassica napus subsp. napus(canola).

Grown on MS agar plates containing 100 mM NaCl, ME09814-01 andME09814-02 transgenic plants showed significantly greater seedling areaand SGI relative to non-transgenic plants. As shown in Table 8-1, theSGI value of T2-generation ME09814-01 seedlings increased by 29% and theSGI value of T2-generation ME09814-02 seedlings increased by 69% ascompared to non-transgenic control seedlings. In the T₃ generation, theSGI increase was 80% for ME09814-01 and 49% for ME09814-02. Thedifferences between transgenic and non-transgenic seedlings arestatistically significant under the t-test, and clearly demonstrate thatthe enhanced tolerance to salt stress was a result of the ectopicexpression of Ceres Clone 965405 in the ME09814 transgenic lines.

TABLE 8-1 Validation assay of ME09814 on salt tolerance in twogenerations SGI* of SGI of pooled % of transgenics non-transgenicst-Test SGI ME Events Avg SE N Avg SE N t-value t_(0.05) increaseME09814- 2.6841 0.2346 37 2.0812 0.1830 34 2.03 1.67 29.0 01T₂ ME09814-2.6985 0.2438 32 1.5942 0.1909 38 3.57 1.67 69.3 02T₂ ME09814- 3.06640.2934 29 1.6996 0.1724 42 4.02 1.67 80.4 01T₃ ME09814- 2.6878 0.2350 361.8087 0.1743 34 3.00 1.67 48.6 02T₃ *SGI (Salt Growth Index) = seedlingarea × Fv/Fm (photosynthesis efficiency)

Summary of Results:

-   -   Ectopic expression of Ceres Clone 965405 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.    -   The protein encoded by Ceres Clone 965405 is an unknown protein.

EXAMPLE 9 ME07361 (Ceres Clone 5367; SEO ID NO: 245)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 5367. Ceres Clone 5367is a functional homolog of Ceres clone 965405.

Grown on MS agar plates containing 100 mM NaCl, ME07361-04 transgenicplants showed significantly greater seedling area and SGI relative tonon-transgenic plants. As shown in Table 9-1, the SGI value ofT2-generation ME07361-03 seedlings increased by 30.34% and the SGI valueof T2-generation ME07361-04 seedlings increased by 52% as compared tonon-transgenic control seedlings. The differences between transgenic andnon-transgenic seedlings are statistically significant under the t-test,and clearly demonstrate that the enhanced tolerance to salt stress was aresult of the ectopic expression of Ceres Clone 5367 in the ME07361transgenic lines.

TABLE 9-1 Results of ME07361 on salt tolerance assay in T2 generationsSGI* of SGI of pooled transgenics non-transgenics t-Test SGI ME EventsAvg SE N Avg SE N p-value increase ME07361-01 0.89 0.103 16 0.89 0.10520 0.489 −0.45% ME07361-02 1.30 0.160 17 1.22 0.132 18 0.357 06.29%ME07361-03 1.58 0.195 21 1.21 0.151 15 0.073 30.34% ME07361-04 1.980.369 15 1.30 0.145 21 0.049 52.00% *SGI (Salt Growth Index) = seedlingarea × Fv/Fm (photosynthesis efficiency)Transgenic plants of ME07361-04 showed significant better tolerance tohigh salt than pooled non-transgenics.

Summary of Results:

-   -   Ectopic expression of Ceres Clone 5367 under the control of the        35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

EXAMPLE 10 ME09594 (Annot ID 566551; SEQ ID NO: 290)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Annot ID 566551. Annot ID 566551is a functional homolog of Ceres clone 965405.

Grown on MS agar plates containing 100 mM NaCl, ME09594-03 transgenicplants showed significantly greater seedling area and SGI relative tonon-transgenic plants. As shown in Table 10-1, the SGI value ofT2-generation ME09594-03 seedlings increased by 60.09% as compared tonon-transgenic control seedlings. The differences between transgenic andnon-transgenic seedlings are statistically significant under the t-test,and clearly demonstrate that the enhanced tolerance to salt stress was aresult of the ectopic expression of Annot ID 566551 in the ME09594transgenic lines.

TABLE 10-1 Results of ME09594 on salt tolerance assay in T2/T3generation SGI* of SGI of pooled transgenics non-transgenics t-Test SGIME Events Avg SE N Avg SE N p-value increase ME09594-01 2.17 0.357 192.36 0.352 16 0.357 −7.87% ME09594- 1.83 1.109 4 1.72 0.252 24 0.4636.20% 02-99 ME09594-03 2.32 0.380 24 1.45 0.280 9 0.038 60.09% ME09594-0.71 0.110 16 0.79 0.082 17 0.288 −9.82% 04-99 ME09594-05 2.38 0.465 132.69 0.392 21 0.305 −11.66% *SGI (Salt Growth Index) = seedling area ×Fv/Fm (photosynthesis efficiency)Transgenic plants of ME09594-03 showed significant better tolerance tohigh salt than pooled non-transgenics.

Summary of Results:

Ectopic expression of Annot ID 566551 under the control of the 35Spromoter enhances tolerance to salt stress that causes necrotic lesionsand stunted growth in wild-type Ws seedlings.

EXAMPLE 11 ME23428 (Annot ID 842118; SEQ ID NO: 289)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Annot ID 842118. Annot ID 842118is a functional homolog of Ceres clone 29658.

Grown on MS agar plates containing 100 mM NaCl, ME23428-02 transgenicplants showed significantly greater seedling area and SGI relative tonon-transgenic plants. As shown in Table 11-1, the SGI value ofT2-generation ME23428-02 seedlings increased by 81.77% as compared tonon-transgenic control seedlings. The differences between transgenic andnon-transgenic seedlings are statistically significant under the t-test,and clearly demonstrate that the enhanced tolerance to salt stress was aresult of the ectopic expression of Annot ID 842118 in the ME23428transgenic lines.

TABLE 11-1 Results of ME23428 on salt tolerance assay in T2 generationSGI* of SGI of pooled transgenics non-transgenics t-Test SGI ME EventsAvg SE N Avg SE N p-value increase ME23428-01 1.26 0.166 9 1.76 0.283 260.069 −28.36% ME23428-02 1.17 0.134 18 0.65 0.139 11 0.005 81.77%ME23428-03 0.63 0.036 13 0.64 0.039 19 0.386 −2.43% ME23428-04 0.930.108 18 0.84 0.222 13 0.371 9.72% ME23428-05 0.99 0.144 19 0.97 0.16614 0.466 1.96% *SGI (Salt Growth Index) = seedling area × Fv/Fm(photosynthesis efficiency)

Transgenic plants of ME23428-02 showed significant better tolerance tohigh salt than pooled non-transgenics.

Summary of Results:

Ectopic expression of Annot ID 842118 under the control of the 35Spromoter enhances tolerance to salt stress that causes necrotic lesionsand stunted growth in wild-type Ws seedlings.

EXAMPLE 12 ME24903 (Clone 295570; SEQ ID NO: 275)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres clone 295570. Ceres clone295570 is a functional homolog of Ceres clone 8686.

Grown on MS agar plates containing 100 mM NaCl, ME24903-04, ME24903-05ME24903-07 and ME24903-09 transgenic plants showed significantly greaterseedling area and SGI relative to non-transgenic plants. As shown inTable 12-1, the SGI value of T2-generation ME24903-04 seedlingsincreased by 68.42%, the SGI value of T2-generation ME24903-05 seedlingsincreased by 55.99%, the SGI value of T2-generation ME24903-07 seedlingsincreased by 140.73% and the SGI value of T2-generation ME24903-09seedlings increased by 121.46% as compared to non-transgenic controlseedlings. The differences between transgenic and non-transgenicseedlings are statistically significant under the t-test, and clearlydemonstrate that the enhanced tolerance to salt stress was a result ofthe ectopic expression of Ceres clone 295570 in the ME24903 transgeniclines.

TABLE 12-1 Results of ME24903 on salt tolerance assay in T2 generationSGI* of SGI of pooled transgenics non-transgenics t-Test SGI ME EventsAvg SE N Avg SE N p-value increase ME24903-04 0.97 0.141 19 0.58 0.09115 0.0123 68.42% ME24903-05 1.72 0.221 18 1.10 0.160 17 0.0153 55.99%ME24903-07 1.51 0.229 21 0.63 0.112 12 0.0008 140.73% ME24903-08 0.890.087 18 0.79 0.208 12 0.3241 13.22% ME24903-09 1.86 0.303 14 0.84 0.10420 0.0016 121.46% *SGI (Salt Growth Index) = seedling area × Fv/Fm(photosynthesis efficiency)

Transgenic plants of ME24903-04,-5,-7 and -09 showed significant bettertolerance to high salt than pooled non-transgenics.

Summary of Results:

Ectopic expression of Ceres clone 295570 under the control of the 35Spromoter enhances tolerance to salt stress that causes necrotic lesionsand stunted growth in wild-type Ws seedlings.

EXAMPLE 13 ME10681 (Clone 335348; SEQ ID NO: 314)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres clone 335348. Ceres clone335348 is a functional homolog of Ceres clone 375578.

Grown on MS agar plates containing 100 mM NaCl, ME10681-02, ME10681-04,and ME10681-05 transgenic plants showed significantly greater seedlingarea and SGI relative to non-transgenic plants. As shown in Table 13-1,the SGI value of T2-generation, the SGI value of T2-generationME10681-02 seedlings increased by 119.17%, the SGI value ofT2-generation ME10681-04 seedlings increased by 113.51% and the SGIvalue of T2-generation ME10681-05 seedlings increased by 103.98% ascompared to non-transgenic control seedlings. The differences betweentransgenic and non-transgenic seedlings are statistically significantunder the t-test, and clearly demonstrate that the enhanced tolerance tosalt stress was a result of the ectopic expression of Ceres clone 335348in the ME10681transgenic lines.

TABLE 13-1 Results of ME10681 on salt tolerance assay in T2 generationSGI* of SGI of pooled transgenics non-transgenics t-Test SGI ME EventsAvg SE N Avg SE N p-value increase ME10681-01 3.87 0.6837 9 2.78 0.432424 0.0940 39.17% ME10681-02 4.13 0.3354 25 1.89 0.5752 11 0.0009 119.17%ME10681-04 6.22 0.4787 12 2.91 0.5671 15 7.66E−05 113.51% ME10681-055.25 0.3916 20 2.57 0.6140 15 0.0004 103.98% *SGI (Salt Growth Index) =seedling area × Fv/Fm (photosynthesis efficiency)

Transgenic plants of ME10681-02,-04 and -05 showed significant bettertolerance to high salt than pooled non-transgenics.

Summary of Results:

Ectopic expression of Ceres clone 335348 under the control of the 35Spromoter enhances tolerance to salt stress that causes necrotic lesionsand stunted growth in wild-type Ws seedlings.

EXAMPLE 14 Determination of Functional Homolog Sequences

The sequences described in the above Examples are utilized as querysequences to identify functional homologs of the query sequences and,together with those sequences, are utilized to define consensussequences for a given group of query and functional homolog sequences.Query sequences and their corresponding functional homolog sequences arealigned to illustrate conserved amino acids consensus sequences thatcontain frequently occurring amino acid residues at particular positionsin the aligned sequences, as shown in FIGS. 1-9.

A subject sequence is considered a functional homolog of a querysequence if the subject and query sequences encode proteins having asimilar function and/or activity. A process known as Reciprocal BLAST(Rivera et al. (1998) Proc. Natl Acad. Sci. USA 95:6239-6244) is used toidentify potential functional homolog sequences from databasesconsisting of all available public and proprietary peptide sequences,including NR from NCBI and peptide translations from Ceres clones.

Before starting a Reciprocal BLAST process, a specific query polypeptideis searched against all peptides from its source species using BLAST inorder to identify polypeptides having sequence identity of 80% orgreater to the query polypeptide and an alignment length of 85% orgreater along the shorter sequence in the alignment. The querypolypeptide and any of the aforementioned identified polypeptides aredesignated as a cluster.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a query polypeptide sequence, “polypeptide A,” from source species S^(A)is BLASTed against all protein sequences from a species of interest. Tophits are determined using an E-value cutoff of 10⁻⁵ and an identitycutoff of 35%. Among the top hits, the sequence having the lowestE-value is designated as the best hit, and considered a potentialfunctional homolog. Any other top hit that had a sequence identity of80% or greater to the best hit or to the original query polypeptide isconsidered a potential functional homolog as well. This process isrepeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species are used to perform a BLAST search against allprotein or polypeptide sequences from the source species S^(A). A tophit from the forward search that returned a polypeptide from theaforementioned cluster as its best hit is also considered as a potentialfunctional homolog.

Functional homologs are identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs areshown in FIGS. 1-9. The Figures represents a grouping of a querysequence aligned with the corresponding identified functional homologsubject sequences. Query sequences and their corresponding functionalhomolog sequences are aligned to identify conserved amino acids and todetermine a consensus sequence that contains a frequently occurringamino acid residue at particular positions in the aligned sequences, asshown in FIGS. 1-9.

An HMM was made based on SEQ ID NOs: 80, 84, 85, 90, 92, 93 and 95,aligned in FIG. 1. When fit to the HMM, SEQ ID NOs: 80, 81, 82, 83, 84,85, 86, 87, 89, 90, 91, 92, 93, 95, 97, 182, 184, 186, 188, 190, 191,and 192 gave HMM bit scores of 576.8, 394.8, 231.4, 382.2, 523.7, 632.7,39.3, 409.6, 386.4, 569.7, 551.4, 621.4, 635.3, 633.5, 573.9, 543.4,594.6546.7, 493.1, 613.4, and 635.3, respectively.

An HMM was made based on SEQ ID NOs: 100, 252, 298, 301, 302, 303 and312 aligned in FIG. 2. When fit to the HMM, SEQ ID NOs: 100, 102, 103,104, 252, 298, 300, 301, 302, 303 and 312 gave HMM bit scores of 1315.8,208.1, 118.5, 173.9, 1272.1, 1235.9, 635.2, 1206.4, 225.6, 1212.9 and1233.4, respectively.

An HMM was made based on SEQ ID NOs: 106, 107, 112, 113, 114 and 115,aligned in FIG. 3. When fit to the HMM, SEQ ID NOs: 106, 107, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 194, 196, 197, 198 and200 gave HMM bit scores of 593.7, 487.6, 238.4, 113.8, 492.6, 536.1,524.8, 289.4, 624.9, 288.2, 476.4, 282.4, 489.3, 588.8, 545.8, 503.3,491.5, 486, 504.9, respectively.

An HMM was made based on SEQ ID NOs: 123, 125, 126, 127, 128, 129 and130, aligned in FIG. 4. When fit to the HMM, SEQ ID NOs: 123, 125, 126,127, 128, 129. 130, 270 and 284 gave HMM bit scores of 390.1, 327.9,392.3, 396.5, 394.8, 393.7, 323.8, 330.6 and 235.5, respectively.

An HMM was made based on SEQ ID NOs: 132, 134, 139, 142 and 143, alignedin FIG. 5. When fit to the HMM, SEQ ID NOs: 132, 134, 136, 138, 139,141, 142, 143 and 144 gave HMM bit scores of 343.8, 454.5, 208, 197,388.2, 144.1, 319.9, 375.2 and 295.2, respectively.

An HMM was made based on SEQ ID NOs: 146, 147, 149, 151 and 152, alignedin FIG. 6. When fit to the HMM, SEQ ID NOs: 146, 147, 149, 150, 151,152, 202, 204 and 206 gave HMM bit scores of 593.1, 602.9, 570.8, 355.3,633.9, 570.8, 369.3, 474.7 and 357.4, respectively.

An HMM was made based on SEQ ID NOs: 154, 157, 160, 161, 163, 164, 168,and 169, aligned in FIG. 7. When fit to the HMM, SEQ ID NOs: 154, 155,157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 274,278, 282, 286 and 288 gave HMM bit scores of 894.1, 719.9, 901.3, 801.4,747, 810.2, 692.7, 748.7, 779.9, 656.3, 603.6, 485.1, 816.9, 634.5, 149,498, 510, 584.3, 455.2 and 670.6, respectively.

An HMM was made based on SEQ ID NOs: 172, 173, 174, 175, 176, 177 and179, aligned in FIG. 8. When fit to the HMM, SEQ ID NOs: 172, 173, 174,175, 176, 177, 179, 208, 210, 212 and 213 gave HMM bit scores of 533.9,542, 570.8, 559.9, 547.5, 474.8, 531.3, 414.3, 447.1, 358.5 and 344,respectively.

An HMM was made based on SEQ ID NOs: 304, 305, 306, 307, 308, 309, 310and 311, aligned in FIG. 9. When fit to the HMM, SEQ ID NOs: 100, 102,103, 104, 252, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311 and 312 gave HMM bit scores of 418.9, 208.1, 118.5, 173.9,407.6, 490.5, 156.9, 461, 462, 469.6, 462, 461, 406.7, 462, 469.6,418.9, 490.5 and 493.3, 493.3 respectively.

Useful polypeptides of the invention include each of the sequences andcorresponding functional homolog sequences shown in the Figures and/orthe Sequence Listing, as well as polypeptides belonging to thecorresponding consensus sequence families as delineated by HMMs. Indifferent embodiments, consensus sequence families have HMM bit scorelower limits as about 50%, 60%, 70%, 80%, 90%, or 95% of any of the HMMbit scores of the family members presented in this application. In someembodiments the lower HMM bit score limits correspond approximately tothe HMM bit score of any of the family members disclosed in thisapplication. A sequence that has an HMM bit score of 20 means that ithas a 95% likelihood of belonging to the consensus sequence defined by aparticular HMM. Alternative HMM bit scores that are useful for thecurrent invention are 50, 75, 100, 125, 150, 200, 250, 300, 350, 400,450 and 500.

The present invention further encompasses nucleotides that encode theabove described polypeptides, as well as the complements thereof, andincluding alternatives thereof based upon the degeneracy of the geneticcode.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

The following references are cited in the Specification. Each of thereferences from the patent and periodical literature cited herein ishereby expressly incorporated in its entirety by such citation.

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1-13. (canceled)
 14. A food or feed product comprising a tissue from atransgenic plant, wherein the transgenic plant and said tissue comprisea recombinant nucleic acid molecule, wherein the recombinant nucleicacid molecule comprises: (a) a nucleotide sequence that encodes aprotein comprising the amino acid sequence of SEQ ID NO:154, and whereinexpression of the nucleotide sequence in a plant increases salt stresstolerance as compared to a control plant of the same species lackingsaid nucleotide sequence; (b) a nucleotide sequence comprising thepolynucleotide sequence of SEQ ID NO:153, wherein the polynucleotidesequence encodes the protein of SEQ ID NO:154; or (c) a nucleotidesequence hybridizing to the full length complementary sequence of thenucleotide sequence as set forth in SEQ ID NO: 153 under high stringencyconditions which comprise (i) hybridization at 42° C. in 50% formamide,6×SSC or 6×SSPE, 0.05% Blotto or 5×Denhardt's Reagent, 100 μg/mldenatured salmon sperm DNA, 0.05% SDS and washing at 65 ° C. first in2×SSC, 0.1% SDS for at least 30 min to one hour and subsequently in0.1×SSC, 0.5% SDS for at least 30 min to one hour; or (ii) hybridizationat 65° C. in 6×SSC or 6×SSPE, 0.05% Blotto or 5×Denhardt's Reagent, 100μg/ml denatured salmon sperm DNA, 0.05% SDS and washing at 65° C.firstin 2×SSC, 0.1% SDS for at least 30 min to one hour, and subsequently in0.1×SSC, 0.5% SDS for at least 30 min to one hour and wherein saidhybridizing nucleotide sequence encodes a protein which has thefunctional activity of SEQ ID NO: 154; wherein said nucleotide sequenceis operably linked to a heterologous promoter; wherein the proteinencoded by the nucleotide sequence is expressed in the transgenic plant;and wherein expression of the protein encoded by said nucleotidesequence in the transgenic plant increases salt stress tolerance ascompared to a control plant of the same species that does not comprisethe recombinant nucleic acid molecule.
 15. The food or feed productaccording to claim 14, wherein the recombinant nucleic acid moleculecomprises a nucleotide sequence that encodes a protein comprising theamino acid sequence of SEQ ID NO:154.
 16. A method of making fuel orchemical feedstock comprising converting the food or feed productaccording to claim 14 into fuel or chemical feedstocks, and wherein saidfuel or chemical feedstock comprises said recombinant molecule. 17-19.(canceled)
 20. The food or feed product of claim 14, wherein therecombinant nucleic acid molecule comprises the nucleotide sequence ofSEQ ID NO:153.
 21. The food or feed product of claim 14, wherein saidtissue is vegetative tissue comprising the recombinant nucleic acidmolecule.
 22. The food or feed product of claim 14, wherein theheterologous promoter is selected from the group consisting of YP0092(SEQ ID NO: 38), PT0676 (SEQ ID NO: 12), PT0708 (SEQ ID NO: 17), PT0613(SEQ ID NO: 5), PT0672 (SEQ ID NO: 11), PT0678 (SEQ ID NO: 13), PT0688(SEQ ID NO: 15), PT0837 (SEQ ID NO: 24), a napin promoter, an Arcelin-5promoter, a phaseolin gene promoter, a soybean tryp sin inhibitorpromoter, an ACP promoter, a stearoyl-ACP desaturase gene promoter, asoybean α′ subunit of P-conglycinin promoter, an oleosin promoter, a 15kD zein promoter, a 16 kD zein promoter, a 19 kD zein promoter, a 22 kDzein promoter, a 27 kD zein promoter, a Osgt-1 promoter, a beta-amylasegene promoter, and a barley hordein gene promoter.
 23. The food or feedproduct of claim 14, wherein the heterologous promoter is selected fromthe group consisting of p326 (SEQ ID NO: 76), YP0144 (SEQ ID NO: 55),YP0190 (SEQ ID NO: 59), p13879 (SEQ ID NO: 75), YP0050 (SEQ ID NO: 35),p32449 (SEQ ID NO: 77), 21876 (SEQ ID NO: 1), YP0158 (SEQ ID NO: 57),YP0214 (SEQ ID NO: 61), YP0380 (SEQ ID NO: 70), PT0848 (SEQ ID NO: 26),and PT0633 (SEQ ID NO: 7), a cauliflower mosaic virus (CaMV) 35Spromoter, a mannopine synthase (MAS) promoter, a 1′ or 2′ promoterderived from T-DNA of Agrobacterium tumefaciens, a figwort mosaic virus34S promoter, an actin promoter, and an ubiquitin promoter.
 24. The foodor feed product of claim 14, wherein the heterologous promoter isselected from the group consisting of a ribulose-1,5-bisphosphatecarboxylase (RbcS) promoter, a pine cab6 promoter, a Cab-1 gene promoterfrom wheat, a CAB-1 promoter from spinach, a cab 1R promoter from rice,a pyruvate orthophosphate dikinase (PPDK) promoter from corn, a tobaccoLhcb1*2 promoter, a Arabidopsis thaliana SUC2 sucrose-H+ symporterpromoter, a thylakoid membrane protein promoter from spinach, PT0535(SEQ ID NO: 3), PT0668 (SEQ ID NO: 2), PT0886 (SEQ ID NO: 29), PR0924(SEQ ID NO: 265), YP0144 (SEQ ID NO: 55), YP0380 (SEQ ID NO: 70), andPT0585 (SEQ ID NO: 4).